Nucleic acids encoding engineered meganucleases with recognition sequences found in the human t cell receptor alpha constant region gene

ABSTRACT

Disclosed herein are recombinant meganucleases engineered to recognize and cleave a recognition sequence present in the human T cell receptor alpha constant region gene. The present disclosure further relates to the use of such recombinant meganucleases in methods for producing genetically-modified eukaryotic cells.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.17/016,056, filed Sep. 9, 2020, which is a divisional of U.S.application Ser. No. 15/766,290, filed Apr. 5, 2018, which is a nationalstage filing under 35 U.S.C. § 371 of International Application No.PCT/US2016/055472, filed Oct. 5, 2016, and claims priority to U.S.Provisional Application No. 62/297,445, entitled “EngineeredMeganucleases With Recognition Sequences Found In The Human T CellReceptor Alpha Constant Region Gene,” filed Feb. 19, 2016, and U.S.Provisional Application No. 62/237,382, entitled “EngineeredMeganucleases With Recognition Sequences Found In The Human T CellReceptor Alpha Constant Region Gene,” filed Oct. 5, 2015, thedisclosures of each of which are hereby incorporated by reference intheir entireties.

FIELD OF THE INVENTION

The invention relates to the fields of oncology, cancer immunotherapy,molecular biology and recombinant nucleic acid technology. Inparticular, the invention relates to recombinant meganucleasesengineered to recognize and cleave a recognition sequence found in thehuman T cell receptor alpha constant region gene. The invention furtherrelates to the use of such recombinant meganucleases in methods forproducing genetically-modified eukaryotic cells.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB

This application contains a Sequence Listing which has been submitted inASCII format via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Jan. 28, 2022, is namedP1090.70013US04-SEQ-EPG and is 264,117 bytes in size.

BACKGROUND OF THE INVENTION

T cell adoptive immunotherapy is a promising approach for cancertreatment. This strategy utilizes isolated human T cells that have beengenetically-modified to enhance their specificity for a specific tumorassociated antigen. Genetic modification may involve the expression of achimeric antigen receptor (CAR) or an exogenous T cell receptor to graftantigen specificity onto the T cell. By contrast to exogenous T cellreceptors, chimeric antigen receptors derive their specificity from thevariable domains of a monoclonal antibody. Thus, T cells expressingchimeric antigen receptors (CAR T cells) induce tumor immunoreactivityin a major histocompatibility complex non-restricted manner. To date, Tcell adoptive immunotherapy has been utilized as a clinical therapy fora number of cancers, including B cell malignancies (e.g., acutelymphoblastic leukemia (ALL), B cell non-Hodgkin lymphoma (NHL), andchronic lymphocytic leukemia), multiple myeloma, neuroblastoma,glioblastoma, advanced gliomas, ovarian cancer, mesothelioma, melanoma,and pancreatic cancer.

Despite its potential usefulness as a cancer treatment, adoptiveimmunotherapy with CAR T cells has been limited, in part, by expressionof the endogenous T cell receptor on the cell surface. CAR T cellsexpressing an endogenous T cell receptor may recognize major and minorhistocompatibility antigens following administration to an allogeneicpatient, which can lead to the development of graft-versus-host-disease(GVHD). As a result, clinical trials have largely focused on the use ofautologous CAR T cells, wherein a patient's T cells are isolated,genetically-modified to incorporate a chimeric antigen receptor, andthen re-infused into the same patient. An autologous approach providesimmune tolerance to the administered CAR T cells; however, this approachis constrained by both the time and expense necessary to producepatient-specific CAR T cells after a patient's cancer has beendiagnosed.

Thus, it would be advantageous to develop “off the shelf” CAR T cells,prepared using T cells from a third party donor, that have reducedexpression of the endogenous T cell receptor and do not initiate GVHDupon administration. Such products could be generated and validated inadvance of diagnosis, and could be made available to patients as soon asnecessary. Therefore, a need exists for the development of allogeneicCAR T cells that lack an endogenous T cell receptor in order to preventthe occurrence of GVHD.

Genetic modification of genomic DNA can be performed usingsite-specific, rare-cutting, homing endonucleases (also called“meganucleases”) that are engineered to recognize specific DNA sequencesin a locus of interest. Homing endonucleases are a group ofnaturally-occurring nucleases that recognize 15-40 base pair cleavagesites commonly found in the genomes of plants and fungi. They arefrequently associated with parasitic DNA elements, such as group 1self-splicing introns and inteins. They naturally promote homologousrecombination or gene insertion at specific locations in the host genomeby producing a double-stranded break in the chromosome, which recruitsthe cellular DNA-repair machinery (Stoddard (2006), Q. Rev. Biophys.38:49-95). Homing endonucleases are commonly grouped into four families:the LAGLIDADG (SEQ ID NO:7) family, the GIY-YIG family, the His-Cys boxfamily and the HNH family. These families are characterized bystructural motifs, which affect catalytic activity and recognitionsequence. For instance, members of the LAGLIDADG (SEQ ID NO:7) familyare characterized by having either one or two copies of the conservedLAGLIDADG (SEQ ID NO:7) motif (Chevalier et al. (2001), Nucleic AcidsRes. 29(18): 3757-3774). The LAGLIDADG (SEQ ID NO:7) homingendonucleases with a single copy of the LAGLIDADG (SEQ ID NO:7) motifform homodimers, whereas members with two copies of the LAGLIDADG (SEQID NO:7) motif are found as monomers.

Methods for producing engineered, site-specific recombinantmeganucleases are known in the art. I-CreI (SEQ ID NO:6) is a member ofthe LAGLIDADG (SEQ ID NO:7) family of homing endonucleases thatrecognizes and cuts a 22 base pair recognition sequence in thechloroplast chromosome of the algae Chlamydomonas reinhardtii. Geneticselection techniques have been used to modify the wild-type I-CreIcleavage site preference (Sussman et al. (2004), J. Mol. Biol. 342:31-41; Chames et al. (2005), Nucleic Acids Res. 33: e178; Seligman etal. (2002), Nucleic Acids Res. 30: 3870-9, Arnould et al. (2006), J.Mol. Biol. 355: 443-58). More recently, a method of rationally-designingmono-LAGLIDADG (SEQ ID NO:7) homing endonucleases was described that iscapable of comprehensively redesigning I-CreI and other homingendonucleases to target widely-divergent DNA sites, including sites inmammalian, yeast, plant, bacterial, and viral genomes (WO 2007/047859).

As first described in WO 2009/059195, I-CreI and its engineeredderivatives are normally dimeric but can be fused into a singlepolypeptide using a short peptide linker that joins the C-terminus of afirst subunit to the N-terminus of a second subunit (Li et al. (2009),Nucleic Acids Res. 37:1650-62; Grizot et al. (2009), Nucleic Acids Res.37:5405-19.) Thus, a functional “single-chain” meganuclease can beexpressed from a single transcript. Such engineered meganucleasesexhibit an extremely low frequency of off-target cutting. By deliveringa gene encoding a single-chain meganuclease to a cell, it is possible tospecifically and preferentially target, cleave, and disrupt the TCRalpha constant region gene.

The use of engineered meganucleases for cleaving DNA targets in thehuman T cell receptor alpha constant region was previously disclosed inInternational Publication No. WO 2014/191527. The '527 publicationdiscloses variants of the I-OnuI meganuclease that are engineered totarget a recognition sequence (SEQ ID NO:3 of the '527 publication)within exon 1 of the TCR alpha constant region gene. Although the '527publication discusses that a chimeric antigen receptor can be expressedin TCR knockout cells, the authors do not disclose the insertion of thechimeric antigen receptor coding sequence into the meganuclease cleavagesite in the TCR alpha constant region gene.

The use of other nucleases and mechanisms for disrupting expression ofthe endogenous TCR have also been disclosed. For example, the use ofzinc finger nucleases for disrupting TCR genes in human T cells wasdescribed by U.S. Pat. No. 8,95,828 and by U.S. Patent ApplicationPublication No. US2014/034902. U.S. Patent Application Publication No.US2014/0301990 describes the use of zinc finger nucleases andtranscription-activator like effector nucleases (TALENs), and aCRISPR/Cas system with an engineered single guide RNA for targeting TCRgenes in an isolated T cell. U.S. Patent Application Publication No.US2012/0321667 discloses the use of small-hairpin RNAs that targetnucleic acids encoding specific TCRs and/or CD3 chains in T cells.

However, the present invention improves upon the teachings of the priorart. The present inventors are the first to teach genetically-modifiedcells that comprise an exogenous polynucleotide sequence (e.g., achimeric antigen receptor or exogenous TCR coding sequence) insertedinto the human TCR alpha constant region gene, which simultaneouslydisrupts expression of the endogenous T cell receptor at the cellsurface. Further, the prior art does not teach the meganucleases or therecognition sequences described herein, or their use for producing suchgenetically-modified cells.

SUMMARY OF THE INVENTION

The present invention provides recombinant meganucleases that areengineered to recognize and cleave recognition sequences found withinresidues 93-208 of the human T cell receptor (TCR) alpha constant regiongene (SEQ ID NO:1). Such meganuclease are useful for disrupting the TCRalpha constant region gene and, consequently, disrupting the expressionand/or function of the cell surface TCR. Therefore, the meganucleases ofthe invention have utility in immunotherapy, including cancerimmunotherapy. Meganuclease cleavage can disrupt gene function either bythe mutagenic action of non-homologous end joining or by promoting theintroduction of an exogenous polynucleotide into the gene via homologousrecombination. In some embodiments, the introduced exogenouspolynucleotide comprises a nucleic acid sequence encoding a chimericantigen receptor, such that the meganuclease is useful for generating anallogeneic CAR T cell that lacks an endogenous TCR.

The present invention further provides methods comprising the deliveryof a recombinant meganuclease protein, or genes encoding a recombinantmeganuclease, to a eukaryotic cell in order to produce agenetically-modified eukaryotic cell.

Thus, in one aspect, the invention provides a recombinant meganucleasethat recognizes and cleaves a recognition sequence within residues93-208 of the human T cell receptor alpha constant region gene (SEQ IDNO:1). Such a recombinant meganuclease comprises a first subunit and asecond subunit, wherein the first subunit binds to a first recognitionhalf-site of the recognition sequence and comprises a firsthypervariable (HVR1) region, and wherein the second subunit binds to asecond recognition half-site of the recognition sequence and comprises asecond hypervariable (HVR2) region.

In one embodiment, the recognition sequence comprises SEQ ID NO:3 (i.e.,the TRC 1-2 recognition sequence).

In one such embodiment, the first meganuclease subunit comprises anamino acid sequence having at least 80%, at least 85%, at least 90%, orat least 95% sequence identity to residues 198-344 of any one of SEQ IDNOs:8-18 or residues 7-153 of any one of SEQ ID NOs:19-27, and thesecond meganuclease subunit comprises an amino acid sequence having atleast 80%, at least 85%, at least 90%, or at least 95% sequence identityto residues 7-153 of any one of SEQ ID NOs:8-18 or residues 198-344 ofany one of SEQ ID NOs:19-27.

In another such embodiment, the HVR1 region comprises Y at a positioncorresponding to: (a) position 215 of any one of SEQ ID NOs:8-18; or (b)position 24 of any one of SEQ ID NOs:19-27. In another such embodiment,the HVR1 region comprises G at a position corresponding to: (a) position233 of any one of SEQ ID NOs:8-18; or (b) position 42 of any one of SEQID NOs:19-27. In another such embodiment, the HVR1 region comprises oneor more of Y and G at positions corresponding to (a) positions 215 and233, respectively, of any one of SEQ ID NOs:8-18; or (b) positions 24and 42, respectively, of any one of SEQ ID NOs:19-27.

In another such embodiment, the HVR2 region comprises T at a positioncorresponding to: (a) position 26 of any one of SEQ ID NOs:8-18; or (b)position 217 of any one of SEQ ID NOs:19-27. In another such embodiment,the HVR2 region comprises F or Y at a position corresponding to: (a)position 28 of any one of SEQ ID NOs:8-18; or (b) position 219 of anyone of SEQ ID NOs:19-27. In another such embodiment, the HVR2 regioncomprises F at a position corresponding to: (a) position 38 of any oneof SEQ ID NOs:8-18; or (b) position 229 of any one of SEQ ID NOs:19-27.In another such embodiment, the HVR2 region comprises S at a positioncorresponding to: (a) position 44 of any one of SEQ ID NOs:8-18; or (b)position 235 of any one of SEQ ID NOs:19-27. In another such embodiment,the HVR2 region comprises F or Y at a position corresponding to: (a)position 46 of any one of SEQ ID NOs:8-18; or (b) position 237 of anyone of SEQ ID NOs:19-27. In another such embodiment, the HVR2 regioncomprises one or more of T, F or Y, F, S, and F or Y, and R at positionscorresponding to: (a) positions 26, 28, 38, 44, and 46, respectively, ofany one of SEQ ID NOs:8-18; or (b) positions 217, 219, 229, 235, and237, respectively, of any one of SEQ ID NOs:19-27.

In another such embodiment, the HVR1 region comprises residues 215-270of any one of SEQ ID NOs:8-18 or residues 24-79 of any one of SEQ IDNOs:19-27. In another such embodiment, the HVR2 region comprisesresidues 24-79 of any one of SEQ ID NOs:8-18 or residues 215-270 of anyone of SEQ ID NOs:19-27.

In another such embodiment, the first meganuclease subunit comprisesresidues 198-344 of any one of SEQ ID NOs:8-18 or residues 7-153 of anyone of SEQ ID NOs:19-27. In another such embodiment, the secondmeganuclease subunit comprises residues 7-153 of any one of SEQ IDNOs:8-18 or residues 198-344 of any one of SEQ ID NOs:19-27.

In another such embodiment, the recombinant meganuclease is asingle-chain meganuclease comprising a linker, wherein the linkercovalently joins the first subunit and the second subunit.

In another such embodiment, the recombinant meganuclease comprises theamino acid sequence of any one of SEQ ID NOs:8-27.

In a further embodiment, the recognition sequence comprises SEQ ID NO:4(i.e., the TRC 3-4 recognition sequence).

In one such embodiment, the first meganuclease subunit comprises anamino acid sequence having at least 80%, at least 85%, at least 90%, orat least 95% sequence identity to residues 7-153 of SEQ ID NO:28 or 29,and the second meganuclease subunit comprises an amino acid sequencehaving at least 80%, at least 85%, at least 90%, or at least 95%sequence identity to residues 198-344 of SEQ ID NO:28 or 29.

In another such embodiment, the HVR1 region comprises Y at a positioncorresponding to position 24 of SEQ ID NO:28 or 29. In another suchembodiment, the HVR1 region comprises T at a position corresponding toposition 26 of SEQ ID NO:28 or 29. In another such embodiment, the HVR1region comprises Y at a position corresponding to position 46 of SEQ IDNO:28 or 29. In another such embodiment, the HVR1 region comprises oneor more of Y, T, and Y at positions corresponding to positions 24, 26,and 46, respectively, of SEQ ID NO:28 or 29.

In another such embodiment, the HVR2 region comprises H at a positioncorresponding to position 215 of SEQ ID NO:28 or 29. In another suchembodiment, the HVR2 region comprises T at a position corresponding toposition 266 of SEQ ID NO:28 or 29. In another such embodiment, the HVR2region comprises C at a position corresponding to position 268 of SEQ IDNO:28 or 29. In another such embodiment, the HVR2 region comprises oneor more of H, T, and C at positions corresponding to positions 215, 266,and 268 of SEQ ID NOs:28 or 29.

In another such embodiment, the HVR1 region comprises residues 24-79 ofSEQ ID NO:28 or 29. In another such embodiment, the HVR2 regioncomprises residues 215-270 of SEQ ID NO:28 or 29.

In another such embodiment, the first meganuclease subunit comprisesresidues 7-153 of SEQ ID NO:28 or 29. In another such embodiment, thesecond meganuclease subunit comprises residues 198-344 of SEQ ID NO:28or 29.

In another such embodiment, the recombinant meganuclease is asingle-chain meganuclease comprising a linker, wherein the linkercovalently joins the first subunit and the second subunit.

In another such embodiment, the recombinant meganuclease comprises theamino acid sequence of SEQ ID NO:28 or 29.

In a further embodiment, the recognition sequence comprises SEQ ID NO:5(i.e., the TRC 7-8 recognition sequence).

In one such embodiment, the first meganuclease subunit comprises anamino acid sequence having at least 80%, at least 85%, at least 90%, orat least 95% sequence identity to residues 7-153 of SEQ ID NO:30 orresidues 198-344 of SEQ ID NO:31 or 32, and the second meganucleasesubunit comprises an amino acid sequence having at least 80%, at least85%, at least 90%, or at least 95% sequence identity to residues 198-344of SEQ ID NO:30 or residues 7-153 of SEQ ID NO:31 or 32.

In another such embodiment, the HVR1 region comprises Y at a positioncorresponding to: (a) position 24 of SEQ ID NO:30; or (b) position 215of SEQ ID NO:31 or 32.

In another such embodiment, the HVR2 region comprises Y or W at aposition corresponding to: (a) position 215 of SEQ ID NO:30; or (b)position 24 of SEQ ID NO:31 or 32. In another such embodiment, the HVR2region comprises M, L, or W at a position corresponding to: (a) position231 of SEQ ID NO:30; or (b) position 40 of SEQ ID NO:31 or 32. Inanother such embodiment, the HVR2 region comprises Y at a positioncorresponding to: (a) position 237 of SEQ ID NO:30; or (b) position 46of SEQ ID NO:31 or 32. In another such embodiment, the HVR2 regioncomprises one or more of Y or W, M, L, or W, and Y at positionscorresponding to: (a) positions 215, 231, and 237, respectively, of SEQID NO:30; or (b) positions 24, 40, and 46, respectively, of SEQ ID NO:31or 32.

In another such embodiment, the HVR1 region comprises residues 24-79 ofSEQ ID NO:30 or residues 215-270 of SEQ ID NO:31 or 32. In another suchembodiment, the HVR2 region comprises residues 215-270 of SEQ ID NO:30or residues 24-79 of SEQ ID NO:31 or 32.

In another such embodiment, the first meganuclease subunit comprisesresidues 7-153 of SEQ ID NO:30 or residues 198-344 of SEQ ID NO:31 or32. In another such embodiment, the second meganuclease subunitcomprises residues 198-344 of SEQ ID NO:30 or residues 7-153 of SEQ IDNO:31 or 32.

In another such embodiment, the recombinant meganuclease is asingle-chain meganuclease comprising a linker, wherein the linkercovalently joins the first subunit and the second subunit.

In another such embodiment, the recombinant meganuclease comprises theamino acid sequence of any one of SEQ ID NOs:30-32.

In another aspect, the invention provides an isolated polynucleotidecomprising a nucleic acid sequence encoding a recombinant meganucleasedescribed herein.

In the various aspects of the invention, the isolated polynucleotide canbe an mRNA. In one embodiment, the mRNA can encode a single recombinantmeganuclease described herein. In other embodiments, the mRNA can be apolycistronic mRNA (e.g., bicistronic, tricistronic, etc.) comprising acoding sequence for at least one meganuclease described herein and acoding sequence for at least one additional protein (e.g., a secondnuclease). In particular embodiments, a polycistronic mRNA can encodetwo or more meganucleases described herein that target differentrecognition sequences within the same gene (e.g., the T cell receptoralpha constant region gene). In other embodiments, a polycistronic mRNAcan encode a meganuclease described herein and a second nuclease thatrecognizes and cleaves a different recognition sequence within the samegene (e.g., the T cell receptor alpha constant region gene) or,alternatively, recognizes and cleaves a different recognition sequencewithin another gene of interest in the genome. In such embodiments,genetically-modified cells produced using such polycistronic mRNA canhave multiple genes knocked out simultaneously. Further,genetically-modified cells produced using such polycistronic mRNA canhave an exogenous sequence of interest inserted into the genome at oneor more of the cleavage sites produced by the encoded nucleases. Inadditional embodiments, a polycistronic mRNA can encode at least onemeganuclease described herein and one additional protein that isbeneficial to the cell, improves efficiency of insertion of an exogenoussequence of interest into a cleavage site, and/or is beneficial in thetreatment of a disease.

In another aspect, the invention provides a recombinant DNA constructcomprising an isolated polynucleotide, wherein the isolatedpolynucleotide comprises a nucleic acid sequence encoding a recombinantmeganuclease described herein. In one embodiment, the recombinant DNAconstruct encodes a viral vector. In one embodiment, the recombinant DNAconstruct encodes a recombinant AAV vector.

In another aspect, the invention provides a viral vector comprising anisolated polynucleotide, wherein the isolated polynucleotide comprises anucleic acid sequence encoding a recombinant meganuclease describedherein. In another aspect, the invention provides a recombinant AAVvector comprising an isolated polynucleotide, wherein the isolatedpolynucleotide comprises a nucleic acid sequence encoding a recombinantmeganuclease described herein.

In another aspect, the invention provides a method for producing agenetically-modified eukaryotic cell comprising an exogenous sequence ofinterest inserted into a chromosome of the eukaryotic cell, the methodcomprising transfecting a eukaryotic cell with one or more nucleic acidsincluding: (a) a first nucleic acid sequence encoding a recombinantmeganuclease described herein; and (b) a second nucleic acid sequenceincluding the sequence of interest; wherein the recombinant meganucleaseproduces a cleavage site in the chromosome at a recognition sequencecomprising SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5, and wherein thesequence of interest is inserted into the chromosome at the cleavagesite.

In one embodiment of the method, the second nucleic acid furthercomprises sequences homologous to sequences flanking the cleavage site,and the sequence of interest is inserted at the cleavage site byhomologous recombination.

In another embodiment of the method, the second nucleic acid lackssubstantial homology to the cleavage site, and the sequence of interestis inserted into the chromosome by non-homologous end-joining.

In another embodiment of the method, the eukaryotic cell is a human Tcell or a cell derived therefrom.

In another embodiment of the method, the sequence of interest encodes achimeric antigen receptor. In another embodiment of the method, thesequence of interest encodes an exogenous T cell receptor.

In another embodiment of the method, at least the first nucleic acidsequence is introduced into the cell by an mRNA. In some suchembodiments, the mRNA can be a polycistronic mRNA encoding at least onerecombinant meganuclease described herein and at least one additionalprotein (e.g., a second nuclease).

In another embodiment of the method, at least the second nucleic acidsequence is introduced into the eukaryotic cell by a viral vector. Inanother embodiment of the method, the first nucleic acid sequence andthe second nucleic acid sequence are introduced into the eukaryotic cellby the same viral vector or, alternatively, by separate viral vectors.

In another embodiment of the method, at least the second nucleic acidsequence is introduced into the eukaryotic cell by a recombinant AAVvector. In another embodiment of the method, the first nucleic acidsequence and the second nucleic acid sequence are introduced into theeukaryotic cell by the same recombinant AAV vector or, alternatively, byseparate recombinant AAV vectors.

In another embodiment of the method, at least the second nucleic acidsequence is introduced into the eukaryotic cell using a single-strandedDNA template.

In a particular embodiment of the method, the first nucleic acidsequence encoding a recombinant meganuclease described herein isintroduced into the cell by an mRNA, and the second nucleic acidsequence including the exogenous sequence of interest is introduced intothe cell using a viral vector, preferably a recombinant AAV vector,wherein the cell is a human T cell, and wherein the sequence of interestencodes a chimeric antigen receptor. In such an embodiment, the methodproduces a genetically-modified T cell that comprises a chimeric antigenreceptor and that has reduced cell-surface expression of the endogenousT cell receptor when compared to a control cell.

In another aspect, the invention provides a method for producing agenetically-modified eukaryotic cell comprising an exogenous sequence ofinterest inserted into a chromosome of the eukaryotic cell, the methodcomprising: (a) introducing a recombinant meganuclease described hereininto a eukaryotic cell; and (b) transfecting the eukaryotic cell with anucleic acid including the sequence of interest; wherein the recombinantmeganuclease produces a cleavage site in the chromosome at a recognitionsequence comprising SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5, andwherein the sequence of interest is inserted into the chromosome at thecleavage site.

In one embodiment of the method, the nucleic acid further comprisessequences homologous to sequences flanking the cleavage site, and thesequence of interest is inserted at the cleavage site by homologousrecombination.

In another embodiment of the method, the nucleic acid lacks substantialhomology to the cleavage site, and the sequence of interest is insertedinto the chromosome by non-homologous end-joining.

In another embodiment of the method, the eukaryotic cell is a human Tcell or a cell derived therefrom.

In another embodiment of the method, the sequence of interest encodes achimeric antigen receptor. In another embodiment of the method, thesequence of interest encodes an exogenous T cell receptor.

In another embodiment of the method, the nucleic acid sequence isintroduced into the eukaryotic cell by a viral vector.

In another embodiment of the method, the nucleic acid sequence isintroduced into the eukaryotic cell by a recombinant AAV vector.

In another embodiment of the method, the nucleic acid sequence isintroduced into the eukaryotic cell using a single-stranded DNAtemplate.

In another aspect, the invention provides a method for producing agenetically-modified eukaryotic cell by disrupting a target sequence ina chromosome of the eukaryotic cell, the method comprising: transfectinga eukaryotic cell with a nucleic acid encoding a recombinantmeganuclease described herein; wherein the recombinant meganucleaseproduces a cleavage site in the chromosome at a recognition sequencecomprising SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5, and wherein thetarget sequence is disrupted by non-homologous end-joining at thecleavage site.

In one embodiment of the method, the eukaryotic cell is a human T cellor a cell derived therefrom.

In another embodiment of the method, the nucleic acid encoding therecombinant meganuclease is an mRNA. In some such embodiments, the mRNAcan be a polycistronic mRNA encoding at least one recombinantmeganuclease described herein and at least one additional protein (e.g.,a second nuclease).

In another embodiment, the method further comprises transfecting theeukaryotic cell with a second nucleic acid including an exogenoussequence of interest. In one such embodiment of the method, the secondnucleic acid further comprises sequences homologous to sequencesflanking the cleavage site, and the sequence of interest is inserted atthe cleavage site by homologous recombination.

In another embodiment of the method, the sequence of interest encodes achimeric antigen receptor. In another embodiment of the method, thesequence of interest encodes an exogenous T cell receptor.

In another embodiment of the method, at least the second nucleic acid isintroduced into the eukaryotic cell by a viral vector.

In another embodiment of the method, at least the second nucleic acid isintroduced into the eukaryotic cell by a recombinant AAV vector.

In another embodiment of the method, at least the second nucleic acidsequence is introduced into the eukaryotic cell using a single-strandedDNA template.

In another aspect, the invention provides a method for producing agenetically-modified eukaryotic cell by disrupting a target sequence ina chromosome of the eukaryotic cell, the method comprising: introducinga recombinant meganuclease described herein into a eukaryotic cell;wherein the meganuclease produces a cleavage site in the chromosome at arecognition sequence comprising SEQ ID NO:3, SEQ ID NO:4, or SEQ IDNO:5, and the target sequence is disrupted by non-homologous end-joiningat the cleavage site.

In one embodiment of the method, the eukaryotic cell is a human T cellor a cell derived therefrom.

In another embodiment, the method further comprises transfecting theeukaryotic cell with a nucleic acid including an exogenous sequence ofinterest.

In one such embodiment of the method, the nucleic acid further comprisessequences homologous to sequences flanking the cleavage site, and thesequence of interest is inserted at the cleavage site by homologousrecombination.

In another such embodiment of the method, the sequence of interestencodes a chimeric antigen receptor. In another such embodiment of themethod, the sequence of interest encodes an exogenous T cell receptor.

In another such embodiment of the method, the nucleic acid is introducedinto the eukaryotic cell by a viral vector.

In another such embodiment of the method, the nucleic acid is introducedinto the eukaryotic cell by a recombinant AAV vector.

In another embodiment of the method, the nucleic acid sequence isintroduced into the eukaryotic cell using a single-stranded DNAtemplate.

In another aspect, the invention provides a method of producing agenetically-modified non-human organism comprising: producing agenetically-modified non-human eukaryotic cell according to the methodsdescribed herein; and (b) growing the genetically-modified non-humaneukaryotic cell to produce the genetically-modified non-human organism.

In one embodiment of the method, the non-human eukaryotic cell isselected from the group consisting of a gamete, a zygote, a blastocystcell, an embryonic stem cell, and a protoplast cell.

In another aspect, the invention provides a genetically-modifiedeukaryotic cell described herein for use as a medicament. The inventionfurther provides the use of a genetically-modified eukaryotic celldescribed herein in the manufacture of a medicament for treating adisease in a subject in need thereof. In one such aspect, the medicamentis useful in the treatment of cancer.

In some embodiments, the CAR comprises an extracellular antigen-bindingdomain. In some embodiments, the extracellular ligand-binding domain ormoiety can be in the form of a single-chain variable fragment (scFv)derived from a monoclonal antibody, which provides specificity for aparticular epitope or antigen (e.g., an epitope or antigenpreferentially present on the surface of a cell, such as a cancer cellor other disease-causing cell or particle). The scFv can be attached viaa linker sequence. The extracellular ligand-binding domain can bespecific for any antigen or epitope of interest. In some embodiments,the scFv can be humanized. The extracellular domain of a chimericantigen receptor can also comprise an autoantigen (see, Payne et al.(2016), Science 353 (6295): 179-184), which can be recognized byautoantigen-specific B cell receptors on B lymphocytes, thus directing Tcells to specifically target and kill autoreactive B lymphocytes inantibody-mediated autoimmune diseases. Such CARs can be referred to aschimeric autoantibody receptors (CAARs), and their use is encompassed bythe invention.

In another aspect, the invention provides a method of immunotherapy fortreating cancer in a subject in need thereof comprising administering tothe subject a pharmaceutical composition comprising agenetically-modified cell produced by the methods disclosed herein and apharmaceutically acceptable carrier.

In some embodiments, the cancer is selected from the group consisting ofa cancer of carcinoma, lymphoma, sarcoma, blastomas, and leukemia.

In some embodiments, the cancer is selected from the group consisting ofa cancer of B-cell origin, breast cancer, gastric cancer, neuroblastoma,osteosarcoma, lung cancer, melanoma, prostate cancer, colon cancer,renal cell carcinoma, ovarian cancer, rhabdomyo sarcoma, leukemia, andHodgkin's lymphoma. In some embodiments, the cancer of B-cell origin isselected from the group consisting of B-lineage acute lymphoblasticleukemia, B-cell chronic lymphocytic leukemia, and B-cell non-Hodgkin'slymphoma.

The foregoing and other aspects and embodiments of the present inventioncan be more fully understood by reference to the following detaileddescription and claims. Certain features of the invention, which are,for clarity, described in the context of separate embodiments, may alsobe provided in combination in a single embodiment. All combinations ofthe embodiments are specifically embraced by the present invention andare disclosed herein just as if each and every combination wasindividually and explicitly disclosed. Conversely, various features ofthe invention, which are, for brevity, described in the context of asingle embodiment, may also be provided separately or in any suitablesub-combination. All sub-combinations of features listed in theembodiments are also specifically embraced by the present invention andare disclosed herein just as if each and every such sub-combination wasindividually and explicitly disclosed herein. Embodiments of each aspectof the present invention disclosed herein apply to each other aspect ofthe invention mutatis mutandis.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. TRC recognition sequences in the human TRC alpha constant regiongene. A) Each recognition sequence targeted by a recombinantmeganuclease of the invention comprises two recognition half-sites. Eachrecognition half-site comprises 9 base pairs, separated by a 4 base paircentral sequence. The TRC 1-2 recognition sequence (SEQ ID NO:3) spansnucleotides 187-208 of the human T cell alpha constant region (SEQ IDNO:1), and comprises two recognition half-sites referred to as TRC1 andTRC2. The TRC 3-4 recognition sequence (SEQ ID NO:4) spans nucleotides93-114 of the human T cell alpha constant region (SEQ ID NO:1), andcomprises two recognition half-sites referred to as TRC3 and TRC4. TheTRC 7-8 recognition sequence (SEQ ID NO:5) spans nucleotides 118-139 ofthe human T cell alpha constant region (SEQ ID NO:1), and comprises tworecognition half-sites referred to as TRC7 and TRC8. B) The recombinantmeganucleases of the invention comprise two subunits, wherein the firstsubunit comprising the HVR1 region binds to a first recognitionhalf-site (e.g., TRC1, TRC3, or TRC7) and the second subunit comprisingthe HVR2 region binds to a second recognition half-site (e.g., TRC2,TRC4, or TRC8). In embodiments where the recombinant meganuclease is asingle-chain meganuclease, the first subunit comprising the HVR1 regioncan be positioned as either the N-terminal or C-terminal subunit.Likewise, the second subunit comprising the HVR2 region can bepositioned as either the N-terminal or C-terminal subunit.

FIGS. 2A-B. Amino acid alignment of TRC1-binding subunits. A-B) Somerecombinant meganucleases encompassed by the invention comprise onesubunit that binds the 9 base pair TRC1 recognition half-site of SEQ IDNO:3. Amino acid sequence alignments are provided for the TRC1-bindingsubunits (SEQ ID NOs:33-52) of the recombinant meganucleases set forthin SEQ ID NOs:8-27. As shown, the TRC1-binding subunit of SEQ IDNOs:8-18 comprises residues 198-344, whereas the TRC1-binding subunit ofSEQ ID NOs:19-27 comprises residues 7-153. Each TRC1-binding subunitcomprises a 56 amino acid hypervariable region as indicated. Variableresidues within the hypervariable region are shaded, with the mostfrequent amino acids at each position further highlighted; the mostprevalent residues are bolded, whereas the second most prevalent arebolded and italicized. Residues outside of the hypervariable region areidentical in each subunit, with the exception of a Q or E residue atposition 80 or position 271 (see, U.S. Pat. No. 8,021,867). AllTRC1-binding subunits provided in FIG. 2 share at least 90% sequenceidentity to the TRC1-binding subunit (residues 198-344) of the TRC1-2x.87 EE meganuclease (SEQ ID NO:33). Residue numbers shown are thoseof SEQ ID NOs:8-27.

FIGS. 3A-B. Amino acid alignment of TRC2-binding subunits. A-B) Somerecombinant meganucleases encompassed by the invention comprise onesubunit that binds the 9 base pair TRC2 recognition half-site of SEQ IDNO:3. Amino acid sequence alignments are provided for the TRC2-bindingsubunits (SEQ ID NOs:58-77) of the recombinant meganucleases set forthin SEQ ID NOs:8-27. As shown, the TRC2-binding subunit of SEQ IDNOs:8-18 comprises residues 7-153, whereas the TRC2-binding subunit ofSEQ ID NOs:19-27 comprises residues 198-344. Each TRC2-binding subunitcomprises a 56 amino acid hypervariable region as indicated. Variableresidues within the hypervariable region are shaded, with the mostfrequent amino acids at each position further highlighted; the mostprevalent residues are bolded, whereas the second most prevalent arebolded and italicized. Residues outside of the hypervariable region areidentical in each subunit, with the exceptions of a Q or E residue atposition 80 or position 271 (see, U.S. Pat. No. 8,021,867), and an Rresidue at position 139 of meganucleases TRC 1-2x.87 EE, TRC 1-2x.87 QE,TRC 1-2x.87 EQ, TRC 1-2x.87, and TRC 1-2x.163 (shaded grey andunderlined). All TRC2-binding subunits provided in FIG. 3 share at least90% sequence identity to the TRC2-binding subunit (residues 7-153) ofthe TRC 1-2x.87 EE meganuclease (SEQ ID NO:58). Residue numbers shownare those of SEQ ID NOs:8-27.

FIG. 4. Amino acid alignment of TRC3-binding subunits. Some recombinantmeganucleases encompassed by the invention comprise one subunit thatbinds the 9 base pair TRC3 recognition half-site of SEQ ID NO:4. Aminoacid sequence alignments are provided for the TRC3-binding subunits (SEQID NOs:53 and 54) of the recombinant meganucleases set forth in SEQ IDNOs:28 and 29. As shown, the TRC3-binding subunit of SEQ ID NOs:28 and29 comprises residues 7-153. Each TRC3-binding subunit comprises a 56amino acid hypervariable region as indicated. Variable residues withinthe hypervariable region are shaded. Residues outside of thehypervariable region are identical in each subunit, with the exceptionsof a Q or E residue at position 80 (see, U.S. Pat. No. 8,021,867). TheTRC3-binding subunits of the TRC 3-4x.3 and TRC 3-4x.19 meganucleasesshare 97% sequence identity. Residue numbers shown are those of SEQ IDNOs:28 and 29.

FIG. 5. Amino acid alignment of TRC4-binding subunits. Some recombinantmeganucleases encompassed by the invention comprise one subunit thatbinds the 9 base pair TRC4 recognition half-site of SEQ ID NO:4. Aminoacid sequence alignments are provided for the TRC4-binding subunits (SEQID NOs:78 and 79) of the recombinant meganucleases set forth in SEQ IDNOs:28 and 29. As shown, the TRC4-binding subunit of SEQ ID NOs:28 and29 comprises residues 198-344. Each TRC4-binding subunit comprises a 56amino acid hypervariable region as indicated. Variable residues withinthe hypervariable region are shaded. Residues outside of thehypervariable region are identical in each subunit, with the exceptionsof a Q or E residue at position 80 (see, U.S. Pat. No. 8,021,867). TheTRC4-binding subunits of the TRC 3-4x.3 and TRC 3-4x.19 meganucleasesshare 97% sequence identity. Residue numbers shown are those of SEQ IDNOs:28 and 29.

FIGS. 6A-B. Amino acid alignment of TRC7-binding subunits. A-B) Somerecombinant meganucleases encompassed by the invention comprise onesubunit that binds the 9 base pair TRC7 recognition half-site of SEQ IDNO:5. Amino acid sequence alignments are provided for the TRC7-bindingsubunits (SEQ ID NOs:55-57) of the recombinant meganucleases set forthin SEQ ID NOs:30-32. As shown, the TRC7-binding subunit of SEQ ID NO:30comprises residues 7-153, whereas the TRC7-binding subunit of SEQ IDNOs:31 and 32 comprises residues 198-344. Each TRC7-binding subunitcomprises a 56 amino acid hypervariable region as indicated. Variableresidues within the hypervariable region are shaded, with the mostfrequent amino acids at each position further highlighted; the mostprevalent residues are bolded, whereas the second most prevalent arebolded and italicized. Residues outside of the hypervariable region areidentical in each subunit, with the exception of a Q or E residue atposition 80 or position 271 (see, U.S. Pat. No. 8,021,867). AllTRC7-binding subunits provided in FIG. 6 share at least 90% sequenceidentity to the TRC7-binding subunit (residues 7-153) of the TRC 7-8x.7meganuclease (SEQ ID NO:55). Residue numbers shown are those of SEQ IDNOs:30-32.

FIGS. 7A-B. Amino acid alignment of TRC8-binding subunits. A-B) Somerecombinant meganucleases encompassed by the invention comprise onesubunit that binds the 9 base pair TRC8 recognition half-site of SEQ IDNO:5. Amino acid sequence alignments are provided for the TRC8-bindingsubunits (SEQ ID NOs:80-82) of the recombinant meganucleases set forthin SEQ ID NOs:30-32. As shown, the TRC8-binding subunit of SEQ ID NO:30comprises residues 198-344, whereas the TRC8-binding subunit of SEQ IDNOs:31 and 32 comprises residues 7-153. Each TRC8-binding subunitcomprises a 56 amino acid hypervariable region as indicated. Variableresidues within the hypervariable region are shaded, with the mostfrequent amino acids at each position further highlighted; the mostprevalent residues are bolded, whereas the second most prevalent arebolded and italicized. Residues outside of the hypervariable region areidentical in each subunit, with the exception of a Q or E residue atposition 80 or position 271 (see, U.S. Pat. No. 8,021,867). AllTRC8-binding subunits provided in FIG. 7 share at least 90% sequenceidentity to the TRC8-binding subunit (residues 198-344) of the TRC7-8x.7 meganuclease (SEQ ID NO:80). Residue numbers shown are those ofSEQ ID NOs:30-32.

FIG. 8. Schematic of reporter assay in CHO cells for evaluatingrecombinant meganucleases targeting recognition sequences found in the Tcell receptor alpha constant region (SEQ ID NO:1). For the recombinantmeganucleases described herein, a CHO cell line was produced in which areporter cassette was integrated stably into the genome of the cell. Thereporter cassette comprised, in 5′ to 3′ order: an SV40 Early Promoter;the 5′ 2/3 of the GFP gene; the recognition sequence for an engineeredmeganuclease of the invention (e.g., the TRC 1-2 recognition sequence,the TRC 3-4 recognition sequence, or the TRC 7-8 recognition sequence);the recognition sequence for the CHO-23/24 meganuclease(WO/2012/167192); and the 3′ 2/3 of the GFP gene. Cells stablytransfected with this cassette did not express GFP in the absence of aDNA break-inducing agent. Meganucleases were introduced by transductionof plasmid DNA or mRNA encoding each meganuclease. When a DNA break wasinduced at either of the meganuclease recognition sequences, theduplicated regions of the GFP gene recombined with one another toproduce a functional GFP gene. The percentage of GFP-expressing cellscould then be determined by flow cytometry as an indirect measure of thefrequency of genome cleavage by the meganucleases.

FIG. 9. Efficiency of recombinant meganucleases for recognizing andcleaving recognition sequences in the human T cell receptor alphaconstant region (SEQ ID NO:1) in a CHO cell reporter assay. Each of therecombinant meganucleases set forth in SEQ ID NOs:8-32 were engineeredto target the TRC 1-2 recognition sequence (SEQ ID NO:3), the TRC 3-4recognition sequence (SEQ ID NO:4), or the TRC 7-8 recognition sequence(SEQ ID NO:5), and were screened for efficacy in the CHO cell reporterassay. The results shown provide the percentage of GFP-expressing cellsobserved in each assay, which indicates the efficacy of eachmeganuclease for cleaving a TRC target recognition sequence or theCHO-23/24 recognition sequence. A negative control (RHO 1-2 bs) wasfurther included in each assay. A)-C) Meganucleases targeting the TRC1-2 recognition sequence. D) Meganucleases targeting the TRC 3-4recognition sequence. E)-F) Meganucleases targeting the TRC 7-8recognition sequence. G) Variants of the TRC 1-2x.87 meganuclease,wherein the Q at position 271 is substituted with E (TRC 1-2x.87 QE),the Q at position 80 is substituted with E (TRC 1-2x.87 EQ), or the Q atposition 80 and the Q at position 271 are both substituted with E (TRC1-2x.87 EE).

FIG. 10. Time course of recombinant meganuclease efficacy in CHO cellreporter assay. The TRC 1-2x.87 QE, TRC 1-2x.87 EQ, and TRC 1-2x.87 EEmeganucleases were evaluated in the CHO reporter assay, with thepercentage of GFP-expressing cells determined 1, 4, 6, 8, and 12 daysafter introduction of meganuclease-encoding mRNA into the CHO reportercells.

FIG. 11. Analysis of Jurkat cell genomic DNA following transfection withTRC 1-2 meganucleases. At 72 hours post-transfection with mRNA encodingTRC 1-2 meganucleases, genomic DNA was harvested and a T7 endonucleaseassay was performed to estimate genetic modification at the endogenousTRC 1-2 recognition sequence.

FIG. 12. Dose-response of TRC 1-2 meganuclease expression in Jurkatcells on genetic modification at the endogenous TRC 1-2 recognitionsequence. Jurkat cells were transfected with either 3 μg or 1 μg of agiven TRC 1-2 meganuclease mRNA. At 96 hours, genomic DNA was analyzedusing a T7 endonuclease assay.

FIG. 13. Cleavage of TRC 1-2 recognition sequence in human T cells. A)CD3⁺ T cells were stimulated with anti-CD3 and anti-CD28 antibodies for3 days, then electroporated with mRNA encoding the TRC 1-2x.87 EEmeganuclease. Genomic DNA was harvested at 3 days and 7 dayspost-transfection, and analyzed using a T7 endonuclease assay. B) Todetermine whether mutations at the endogenous TRC 1-2 recognitionsequence were sufficient to eliminate surface expression of the T cellreceptor, cells were analyzed by flow cytometry using an anti-CD3antibody. Control cells (transfected with water) and TRC 1-2x.87EE-transfected cells were analyzed at day 3 and day 7 post-transfection,and the percentage of CD3-positive and CD3-negative T cells wasdetermined.

FIG. 14. Nucleic acid sequences of representative deletions that wereobserved at the TRC 1-2 recognition sequence in human T cells followingexpression of TRC 1-2 meganucleases.

FIG. 15. Diagram illustrating sequence elements of recombinant AAVvectors and their use in combination with an engineered nuclease toinsert an exogenous nucleic acid sequence into the endogenous TCR alphaconstant region gene.

FIG. 16. Map of plasmid used to produce the AAV405 vector.

FIG. 17. Map of plasmid used to produce the AAV406 vector.

FIG. 18. Determining the timing of meganuclease mRNA transfection andrecombinant AAV transduction to enhance AAV transduction efficiency.Human CD3⁺ T cells were electroporated with mRNA encoding the TRC1-2x.87 EE meganuclease and at 2, 4, or 8 hours post-transfection, cellswere transduced with a recombinant AAV vector encoding GFP (GFP-AAV). Tcells were analyzed by flow cytometry for GFP expression at 72 hourspost-transduction to determine transduction efficiency.

FIG. 19. Analyzing human T cells for insertion of an exogenous nucleicacid sequence using recombinant AAV vectors. CD3⁺ T cells transfectedwith TRC 1-2x.87 EE mRNA and subsequently transduced (2 hourspost-transfection) with AAV405 or AAV406. Transduction-only controlswere mock transfected (with water) and transduced with either AAV405 orAAV406. Meganuclease-only controls were transfected with TRC 1-2x.87 EEand then mock transduced (with water) at 2 hours post-transfection.Genomic DNA was harvested from T cells and the TRC 1-2 locus wasamplified by PCR using primers that recognized sequences beyond theregion of homology in the AAV vectors. PCR primers outside of thehomology regions only allowed for amplification of the T cell genome,not from the AAV vectors. PCR products were purified and digested withEagI. PCR products were then analyzed for cleavage.

FIG. 20. Characterization of EagI insertion into the TRC 1-2 recognitionsequence of human T cells using AAV405. A) Undigested PCR productgenerated from previous experiments was cloned into a pCR-blunt vector.Colony PCR was performed using M13 forward and reverse primers and aportion of PCR products from cells transfected with TRC 1-2x.87 EE andAAV405 was analyzed by gel electrophoresis. Analysis shows a mix offull-length PCR products (approximately 1600 bp), smaller inserts, andempty plasmids (approximately 300 bp). B) In parallel, another portionof PCR products were digested with EagI to determine the percent ofclones that contain the EagI recognition site inserted into the TRC 1-2recognition sequence. PCR products cleaved with EagI generated expectedfragments of approximately 700 and 800 bp.

FIG. 21. Characterization of EagI insertion into the TRC 1-2 recognitionsequence of human T cells using AAV406. A) Undigested PCR productgenerated from previous experiments was cloned into a pCR-blunt vector.Colony PCR was performed using M13 forward and reverse primers and aportion of PCR products from cells transfected with TRC 1-2x.87 EE andAAV406 was analyzed by gel electrophoresis. Analysis shows a mix offull-length PCR products (approximately 1600 bp), smaller inserts, andempty plasmids (approximately 300 bp). B) In parallel, another portionof PCR products were digested with EagI to determine the percent ofclones that contain the EagI recognition site inserted into the TRC 1-2recognition sequence. PCR products cleaved with EagI generated expectedfragments of approximately 700 and 800 bp.

FIG. 22. A) Nucleic acid sequences of representative deletions andinsertions (i.e., indels) that were observed at the TRC 1-2 recognitionsequence in human T cells following expression of TRC 1-2 meganucleases.B) Nucleic acid sequence of the TRC 1-2 recognition sequence confirminginsertion of the exogenous nucleic acid sequence comprising the EagIrestriction site.

FIG. 23. Enhancement of recombinant AAV transduction efficiency.Transduction efficiency was further analyzed by optimizing the timing ofmeganuclease mRNA transfection and subsequent AAV transduction. HumanCD3⁺ T cells were electroporated with mRNA encoding the TRC 1-2x.87 EEmeganuclease and subsequently transduced with GFP-AAV immediately aftertransfection or 2 hours post-transfection. Additionally, non-stimulatedresting T cells were transduced with GFP-AAV. Mock transduced cells werealso analyzed. At 72 hours post-transduction, cells were analyzed byflow cytometry for GFP expression to determine AAV transductionefficiency.

FIG. 24. Map of plasmid used to produce the AAV-CAR100 (AAV408) vector.

FIG. 25. Map of plasmid used to produce the AAV-CAR763 (AAV412) vector.

FIG. 26. Insertion of chimeric antigen receptor coding sequence at TRC1-2 recognition site in human T cells. A PCR-based assay was developedto determine whether the AAV412 HDR template was utilized to repairdouble-strand breaks at the TRC 1-2 recognition sequence.

FIG. 27. Insertion of chimeric antigen receptor coding sequence at TRC1-2 recognition site in human T cells. A PCR-based assay was developedto determine whether the AAV408 HDR template was utilized to repairdouble-strand breaks at the TRC 1-2 recognition sequence. A) PCRproducts generated using a primer pair that only amplifies a product onthe 5′ end of the TRC 1-2 recognition sequence locus if the CAR gene hasbeen inserted into that locus. B) PCR products generated using a primerpair that only amplifies a product on the 3′ end of the TRC 1-2recognition sequence locus if the CAR gene has been inserted into thatlocus.

FIG. 28. Digital PCR. A) Schematic of a digital PCR assay developed toquantitatively determine insertion efficiency of the chimeric antigenreceptor coding sequence into the TRC 1-2 recognition site in human Tcells. B) Results of digital PCR on genomic DNA from human T cellselectroporated with a TRC 1-2x.87EE meganuclease mRNA and/or increasingamounts of AAV408.

FIG. 29. Cell-surface expression of CD19 chimeric antigen receptor onhuman T cells. The expression level of the anti-CD19 chimeric antigenreceptor was determined in cells that had the CAR gene inserted into theTRC 1-2 recognition sequence using AAV408 as the HDR template.Cell-surface expression was analyzed by flow cytometry. A) Cells thatwere mock electroporated and mock transduced (MOI—0), and cells thatwere mock electroporated and transduced with increasing amounts ofAAV408. B) Cells that were electroporated with TRC 1-2x.87EE and mocktransduced (MOI—0), and cells that were electroporated with TRC1-2x.87EE and transduced with increasing amounts of AAV408.

FIG. 30. Map of plasmid used to produce the AAV421 vector.

FIG. 31. Map of plasmid used to produce the AAV422 vector.

FIG. 32. Insertion of chimeric antigen receptor coding sequence. PCRmethods were used to determine if the chimeric antigen receptor codingsequence introduced by AAV421 or AAV422 inserted at the TRC 1-2recognition site cleaved by the TRC 1-2x.87EE meganuclease. A) Analysisof insertion following transduction with AAV421. B) Analysis ofinsertion following transduction with AAV422.

FIG. 33. Cell-surface expression of CD19 chimeric antigen receptor onhuman T cells. The expression level of the anti-CD19 chimeric antigenreceptor was determined in cells that had the CAR gene inserted into theTRC 1-2 recognition sequence using AAV421 as the HDR template.Cell-surface expression was analyzed by flow cytometry. A) Cells thatwere mock electroporated and mock transduced (MOI—0), and cells thatwere mock electroporated and transduced with increasing amounts ofAAV421. B) Cells that were electroporated with TRC 1-2x.87EE and mocktransduced (MOI—0), and cells that were electroporated with TRC1-2x.87EE and transduced with increasing amounts of AAV421.

FIG. 34. Expansion of human T cells expressing a cell-surface chimericantigen receptor. Methods were determined for preferentially expandingand enriching a CD3⁻/CAR⁺ T cell population following electroporationwith mRNA for the TRC 1-2x.87EE meganuclease and transduction withAAV421. A) Supplementation with IL-7 (10 ng/mL) and IL-15 (10 ng/mL). B)Supplementation with IL-7 (10 ng/mL) and IL-15 (10 ng/mL), andincubation with mitomycin C-inactivated IM-9 cells. C) Supplementationwith IL-7 (10 ng/mL) and IL-15 (10 ng/mL), and two incubations withmitomycin C-inactivated IM-9 cells.

FIG. 35. Cell-surface expression of CD19 chimeric antigen receptor onhuman T cells. The expression level of the anti-CD19 chimeric antigenreceptor was determined in cells that had the CAR gene inserted into theTRC 1-2 recognition sequence using AAV422 as the HDR template.Cell-surface expression was analyzed by flow cytometry. A) Cells thatwere mock electroporated and mock transduced (MOI—0), and cells thatwere mock electroporated and transduced with increasing amounts ofAAV422. B) Cells that were electroporated with TRC 1-2x.87EE and mocktransduced (MOI—0), and cells that were electroporated with TRC1-2x.87EE and transduced with increasing amounts of AAV422.

FIG. 36. Expansion of human T cells expressing a cell-surface chimericantigen receptor. Methods were determined for preferentially expandingand enriching a CD3⁻/CAR⁺ T cell population following electroporationwith mRNA for the TRC 1-2x.87EE meganuclease and transduction withAAV422. A) Supplementation with IL-7 (10 ng/mL) and IL-15 (10 ng/mL). B)Supplementation with IL-7 (10 ng/mL) and IL-15 (10 ng/mL), andincubation with mitomycin C-inactivated IM-9 cells. C) Supplementationwith IL-7 (10 ng/mL) and IL-15 (10 ng/mL), and two incubations withmitomycin C-inactivated IM-9 cells.

FIG. 37. Meganuclease knockout efficiency using single-strand AAV.Experiments were conducted to examine the knockout efficiency of twomeganucleases in human T cells when simultaneously transduced with asingle-stranded AAV vector. A) Cells electroporated with mRNA for TRC1-2x.87EE and transduced with increasing amounts of the single-strandedAAV412. B) Cells electroporated with mRNA for a meganuclease targetingthe beta-2 microglobulin gene and transduced with increasing amounts ofthe single-stranded AAV412. C) Cells electroporated with mRNA for TRC1-2x.87EE and transduced with increasing amounts of the single-strandedAAV422.

FIG. 38. Functional activity of anti-CD19 CAR T cells. A) IFN-gammaELISPOT assay, in which either CD19⁺ Raji cells or CD19⁻ U937 cells werethe target population. B) Cell killing assay in which luciferase-labeledCD19⁺ Raji cells were the target.

FIG. 39. Expression of chimeric antigen receptors following transductionwith linearized DNA donor templates. These experiments generatedplasmids that contain an anti-CD19 CAR gene flanked by homology armsthat are homologous to the TRC 1-2 recognition sequence locus. Differentpromoters were used in some plasmids, and homology arms were either“short” (200 bp on the 5′ homology arm and 180 bp on the 3′ homologyarm) or “long” (985 bp on the 5′ homology arm and 763 bp on the 3′homology arm). CAR donor plasmids were linearized at a restriction sitein the vector backbone and gel purified. A) Background CD3⁻/CAR⁺staining. B) Cells electroporated with TRC 1-2x.87EE mRNA alone. C)Cells co-electroporated with TRC 1-2x.87EE mRNA and a long homology armvector with an EF1α core promoter with an HTLV enhancer. D) Cellsco-electroporated with TRC 1-2x.87EE mRNA and a short homology armvector with EF1α core promoter (with no enhancer). E) Cellselectroporated with a long homology arm vector with an EF1α corepromoter with an HTLV enhancer in the absence of TRC 1-2x.87EE mRNA. F)Cells electroporated with a short homology arm vector with EF1α corepromoter (with no enhancer) in the absence of TRC 1-2x.87EE mRNA. G)Cells electroporated with a long homology arm construct that contains anMND promoter driving expression of the CAR and an intron in the 5′ endof the CAR gene, as well as TRC 1-2x.87EE mRNA. H) Cells electroporatedwith a long homology arm construct that contains an MND promoter drivingexpression of the CAR and no intron, as well as TRC 1-2x.87EE mRNA. I)Cells electroporated with a short homology arm plasmid with the MNDpromoter and no intron, as well as TRC 1-2x.87EE mRNA. J) Cellselectroporated with a long homology arm construct that contains an MNDpromoter driving expression of the CAR and an intron in the 5′ end ofthe CAR gene, but no TRC 1-2x.87EE mRNA. K) Cells electroporated with along homology arm construct that contains an MND promoter drivingexpression of the CAR and no intron, but no TRC 1-2x.87EE mRNA. L) Cellselectroporated with a short homology arm plasmid with the MND promoterand no intron, but no TRC 1-2x.87EE mRNA. M) Cells electroporated with ashort homology arm construct that contained a JeT promoter, as well asTRC 1-2x.87EE mRNA. N) Cells electroporated with a long homology armconstruct that contained a CMV promoter, as well as TRC 1-2x.87EE mRNA.0) Cells electroporated with a short homology arm construct thatcontained a JeT promoter, but no TRC 1-2x.87EE mRNA. P) Cellselectroporated with a long homology arm construct that contained a CMVpromoter, but no TRC 1-2x.87EE mRNA.

FIG. 40. PCR analysis to determine whether the chimeric antigen receptorcoding region delivered by linearized DNA constructs was inserted intothe TRC 1-2 recognition sequence in human T cells.

FIG. 41. Map of plasmid used to produce the AAV423 vector.

FIG. 42. Cell-surface expression of CD19 chimeric antigen receptor onhuman T cells. The expression level of the anti-CD19 chimeric antigenreceptor was determined in cells that had the CAR gene inserted into theTRC 1-2 recognition sequence using AAV423 as the HDR template.Cell-surface expression was analyzed by flow cytometry. A) Cells thatwere mock electroporated and mock transduced (MOI—0), and cells thatwere mock electroporated and transduced with increasing amounts ofAAV423. B) Cells that were electroporated with TRC 1-2x.87EE and mocktransduced (MOI—0), and cells that were electroporated with TRC1-2x.87EE and transduced with increasing amounts of AAV423.

FIG. 43. Insertion of chimeric antigen receptor coding sequence. PCRmethods were used to determine if the chimeric antigen receptor codingsequence introduced by AAV423 inserted at the TRC 1-2 recognition sitecleaved by the TRC 1-2x.87EE meganuclease.

FIG. 44. Phenotype analysis of anti-CD19 CAR T cells. A) Activated Tcells were electroporated with TRC 1-2x.87 EE mRNA, then transduced withan AAV6 vector comprising an anti-CD19 CAR expression cassette driven bya JeT promoter and flanked by homology arms. Following 5 days of culturewith IL-2 (10 ng/mL), cells were analyzed for cell-surface CD3 andanti-CD19 CAR expression by flow cytometry. B) CD3⁻ cells were enrichedby depleting CD3+ cells using anti-CD3 magnetic beads. Depleted cellswere then cultured for 3 days in IL-15 (10 ng/mL) and IL-21 (10 ng/mL)and re-analyzed for cell-surface expression of CD3 and anti-CD19 CAR. C)The purified population of CD3⁻ CD19-CAR T cells was analyzed by flowcytometry to determine the percentage of cells that were CD4⁺ and CD8⁺.D) The purified population of CD3⁻ CD19-CAR T cells was further analyzedby flow cytometry to determine whether they were central memory T cells,transitional memory T cells, or effector memory T cells by staining forCD62L and CD45RO.

FIG. 45. Raji disseminated lymphoma model. Raji cells stably expressingfirefly luciferase (ffLuc)⁴⁴ were injected i.v. into 5-6 week old femaleNSG mice on Day 1, at a dose of 2.0×10⁵ cells per mouse. On Day 4 micewere injected i.v. with PBS or PBS containing gene edited control TCR KOT cells prepared from the same healthy donor PBMC or PBS containing theindicated doses of CAR T cells prepared from the same donor. On theindicated days, live mice were injected i.p. with Luciferin substrate(150 mg/kg in saline), anesthetized, and Luciferase activity measuredafter 7 minutes using IVIS SpectrumCT® (Perkin Elmer, Waltham, Mass.).Data was analyzed and exported using Living Image software 4.5.1 (PerkinElmer, Waltham, Mass.). Luminescence signal intensity is represented byradiance in p/sec/cm²/sr.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 sets forth the nucleotide sequence of the human T cellreceptor alpha constant region gene (NCBI Gene ID NO. 28755).

SEQ ID NO: 2 sets forth the amino acid sequence encoded by the human Tcell receptor alpha constant region.

SEQ ID NO: 3 sets forth the amino acid sequence of the TRC 1-2recognition sequence.

SEQ ID NO: 4 sets forth the nucleotide sequence of the TRC 3-4recognition sequence.

SEQ ID NO: 5 sets forth the nucleotide sequence of the TRC 7-8recognition sequence.

SEQ ID NO: 6 sets forth the amino acid sequence of I-CreI.

SEQ ID NO: 7 sets forth the amino acid sequence of the LAGLIDADG motif.

SEQ ID NO: 8 sets forth the amino acid sequence of the TRC 1-2x.87 EEmeganuclease.

SEQ ID NO: 9 sets forth the amino acid sequence of the TRC 1-2x.87 QEmeganuclease.

SEQ ID NO: 10 sets forth the amino acid sequence of the TRC 1-2x.87 EQmeganuclease.

SEQ ID NO: 11 sets forth the amino acid sequence of the TRC 1-2x.87meganuclease.

SEQ ID NO: 12 sets forth the amino acid sequence of the TRC 1-2x.6meganuclease.

SEQ ID NO: 13 sets forth the amino acid sequence of the TRC 1-2x.20meganuclease.

SEQ ID NO: 14 sets forth the amino acid sequence of the TRC 1-2x.55meganuclease.

SEQ ID NO: 15 sets forth the amino acid sequence of the TRC 1-2x.60meganuclease.

SEQ ID NO: 16 sets forth the amino acid sequence of the TRC 1-2x.105meganuclease.

SEQ ID NO: 17 sets forth the amino acid sequence of the TRC 1-2x.163meganuclease.

SEQ ID NO: 18 sets forth the amino acid sequence of the TRC 1-2x.113_3meganuclease.

SEQ ID NO: 19 sets forth the amino acid sequence of the TRC 1-2x.5meganuclease.

SEQ ID NO: 20 sets forth the amino acid sequence of the TRC 1-2x.8meganuclease.

SEQ ID NO: 21 sets forth the amino acid sequence of the TRC 1-2x.25meganuclease.

SEQ ID NO: 22 sets forth the amino acid sequence of the TRC 1-2x.72meganuclease.

SEQ ID NO: 23 sets forth the amino acid sequence of the TRC 1-2x.80meganuclease.

SEQ ID NO: 24 sets forth the amino acid sequence of the TRC 1-2x.84meganuclease.

SEQ ID NO: 25 sets forth the amino acid sequence of the TRC 1-2x.120meganuclease.

SEQ ID NO: 26 sets forth the amino acid sequence of the TRC 1-2x.113_1meganuclease.

SEQ ID NO: 27 sets forth the amino acid sequence of the TRC 1-2x.113_2meganuclease.

SEQ ID NO: 28 sets forth the amino acid sequence of the TRC 3-4x.3meganuclease.

SEQ ID NO: 29 sets forth the amino acid sequence of the TRC 3-4x.19meganuclease.

SEQ ID NO: 30 sets forth the amino acid sequence of the TRC 7-8x.7meganuclease.

SEQ ID NO: 31 sets forth the amino acid sequence of the TRC 7-8x.9meganuclease.

SEQ ID NO: 32 sets forth the amino acid sequence of the TRC 7-8x.14meganuclease.

SEQ ID NO: 33 sets forth residues 198-344 of the TRC 1-2x.87 EEmeganuclease.

SEQ ID NO: 34 sets forth residues 198-344 of the TRC 1-2x.87 QEmeganuclease.

SEQ ID NO: 35 sets forth residues 198-344 of the TRC 1-2x.87 EQmeganuclease.

SEQ ID NO: 36 sets forth residues 198-344 of the TRC 1-2x.87meganuclease.

SEQ ID NO: 37 sets forth residues 198-344 of the TRC 1-2x.6meganuclease.

SEQ ID NO: 38 sets forth residues 198-344 of the TRC 1-2x.20meganuclease.

SEQ ID NO: 39 sets forth residues 198-344 of the TRC 1-2x.55meganuclease.

SEQ ID NO: 40 sets forth residues 198-344 of the TRC 1-2x.60meganuclease.

SEQ ID NO: 41 sets forth residues 198-344 of the TRC 1-2x.105meganuclease.

SEQ ID NO: 42 sets forth residues 198-344 of the TRC 1-2x.163meganuclease.

SEQ ID NO: 43 sets forth residues 198-344 of the TRC 1-2x.113_3meganuclease.

SEQ ID NO: 44 sets forth residues 7-153 of the TRC 1-2x.5 meganuclease.

SEQ ID NO: 45 sets forth residues 7-153 of the TRC 1-2x.8 meganuclease.

SEQ ID NO: 46 sets forth residues 7-153 of the TRC 1-2x.25 meganuclease.

SEQ ID NO: 47 sets forth residues 7-153 of the TRC 1-2x.72 meganuclease.

SEQ ID NO: 48 sets forth residues 7-153 of the TRC 1-2x.80 meganuclease.

SEQ ID NO: 49 sets forth residues 7-153 of the TRC 1-2x.84 meganuclease.

SEQ ID NO: 50 sets forth residues 7-153 of the TRC 1-2x.120meganuclease.

SEQ ID NO: 51 sets forth residues 7-153 of the TRC 1-2x.113_1meganuclease.

SEQ ID NO: 52 sets forth residues 7-153 of the TRC 1-2x.113_2meganuclease.

SEQ ID NO: 53 sets forth residues 7-153 of the TRC 3-4x.3 meganuclease.

SEQ ID NO: 54 sets forth residues 7-153 of the TRC 3-4x.19 meganuclease.

SEQ ID NO: 55 sets forth residues 7-153 of the TRC 7-8x.7 meganuclease.

SEQ ID NO: 56 sets forth residues 198-344 of the TRC 7-8x.9meganuclease.

SEQ ID NO: 57 sets forth residues 198-344 of the TRC 7-8x.14meganuclease.

SEQ ID NO: 58 sets forth residues 7-153 of the TRC 1-2x.87 EEmeganuclease.

SEQ ID NO: 59 sets forth residues 7-153 of the TRC 1-2x.87 QEmeganuclease.

SEQ ID NO: 60 sets forth residues 7-153 of the TRC 1-2x.87 EQmeganuclease.

SEQ ID NO: 61 sets forth residues 7-153 of the TRC 1-2x.87 meganuclease.

SEQ ID NO: 62 sets forth residues 7-153 of the TRC 1-2x.6 meganuclease.

SEQ ID NO: 63 sets forth residues 7-153 of the TRC 1-2x.20 meganuclease.

SEQ ID NO: 64 sets forth residues 7-153 of the TRC 1-2x.55 meganuclease.

SEQ ID NO: 65 sets forth residues 7-153 of the TRC 1-2x.60 meganuclease.

SEQ ID NO: 66 sets forth residues 7-153 of the TRC 1-2x.105meganuclease.

SEQ ID NO: 67 sets forth residues 7-153 of the TRC 1-2x.163meganuclease.

SEQ ID NO: 68 sets forth residues 7-153 of the TRC 1-2x.113_3meganuclease.

SEQ ID NO: 69 sets forth residues 198-344 of the TRC 1-2x.5meganuclease.

SEQ ID NO: 70 sets forth residues 198-344 of the TRC 1-2x.8meganuclease.

SEQ ID NO: 71 sets forth residues 198-344 of the TRC 1-2x.25meganuclease.

SEQ ID NO: 72 sets forth residues 198-344 of the TRC 1-2x.72meganuclease.

SEQ ID NO: 73 sets forth residues 198-344 of the TRC 1-2x.80meganuclease.

SEQ ID NO: 74 sets forth residues 198-344 of the TRC 1-2x.84meganuclease.

SEQ ID NO: 75 sets forth residues 198-344 of the TRC 1-2x.120meganuclease.

SEQ ID NO: 76 sets forth residues 198-344 of the TRC 1-2x.113_1meganuclease.

SEQ ID NO: 77 sets forth residues 198-344 of the TRC 1-2x.113_2meganuclease.

SEQ ID NO: 78 sets forth residues 198-344 of the TRC 3-4x.3meganuclease.

SEQ ID NO: 79 sets forth residues 198-344 of the TRC 3-4x.19meganuclease.

SEQ ID NO: 80 sets forth residues 198-344 of the TRC 7-8x.7meganuclease.

SEQ ID NO: 81 sets forth residues 7-153 of the TRC 7-8x.9 meganuclease.

SEQ ID NO: 82 sets forth residues 7-153 of the TRC 7-8x.14 meganuclease.

SEQ ID NO: 83 sets forth the nucleotide sequence of the antisense strandof the TRC 1-2 recognition sequence.

SEQ ID NO: 84 sets forth the nucleotide sequence of the antisense strandof the TRC 3-4 recognition sequence.

SEQ ID NO: 85 sets forth the nucleotide sequence of the antisense strandof the TRC 7-8 recognition sequence.

SEQ ID NO: 86 sets forth nucleotides 162-233 of SEQ ID NO:1.

SEQ ID NO: 87 sets forth nucleotides 162-233 of SEQ ID NO:1 comprising adeletion resulting from cleavage and NHEJ.

SEQ ID NO: 88 sets forth nucleotides 162-233 of SEQ ID NO:1 comprising adeletion resulting from cleavage and NHEJ.

SEQ ID NO: 89 sets forth nucleotides 162-233 of SEQ ID NO:1 comprising adeletion resulting from cleavage and NHEJ.

SEQ ID NO: 90 sets forth nucleotides 162-233 of SEQ ID NO:1 comprising adeletion resulting from cleavage and NHEJ.

SEQ ID NO: 91 sets forth nucleotides 162-233 of SEQ ID NO:1 comprising adeletion resulting from cleavage and NHEJ.

SEQ ID NO: 92 sets forth nucleotides 162-233 of SEQ ID NO:1 comprising adeletion resulting from cleavage and NHEJ.

SEQ ID NO: 93 sets forth nucleotides 162-233 of SEQ ID NO:1 comprising adeletion resulting from cleavage and NHEJ.

SEQ ID NO: 94 sets forth nucleotides 162-233 of SEQ ID NO:1 comprisingan insertion resulting from cleavage and NHEJ.

SEQ ID NO: 95 sets forth nucleotides 162-233 of SEQ ID NO:1 comprisingan insertion resulting from cleavage and NHEJ.

SEQ ID NO: 96 sets forth nucleotides 162-233 of SEQ ID NO:1 comprising adeletion resulting from cleavage and NHEJ.

SEQ ID NO: 97 sets forth nucleotides 162-233 of SEQ ID NO:1 comprising adeletion resulting from cleavage and NHEJ.

SEQ ID NO: 98 sets forth nucleotides 162-233 of SEQ ID NO:1 comprising adeletion resulting from cleavage and NHEJ.

SEQ ID NO: 99 sets forth nucleotides 162-233 of SEQ ID NO:1 comprising adeletion resulting from cleavage and NHEJ.

SEQ ID NO: 100 sets forth nucleotides 162-233 of SEQ ID NO:1 comprisinga deletion resulting from cleavage and NHEJ.

SEQ ID NO: 101 sets forth nucleotides 162-233 of SEQ ID NO:1 comprisinga deletion resulting from cleavage and NHEJ.

SEQ ID NO: 102 sets forth nucleotides 162-233 of SEQ ID NO:1 comprisinga deletion resulting from cleavage and NHEJ.

SEQ ID NO: 103 sets forth nucleotides 162-233 of SEQ ID NO:1 comprisinga deletion resulting from cleavage and NHEJ.

SEQ ID NO: 104 sets forth nucleotides 162-233 of SEQ ID NO:1 comprisinga deletion resulting from cleavage and NHEJ.

SEQ ID NO: 105 sets forth nucleotides 181-214 of SEQ ID NO:1.

SEQ ID NO: 106 sets forth nucleotides 181-214 of SEQ ID NO:1 comprisingan exogenous nucleic acid sequence inserted via homologousrecombination.

SEQ ID NO: 107 sets forth the nucleotide sequence of a plasmid used togenerate the AAV405 vector.

SEQ ID NO: 108 sets forth the nucleotide sequence of a plasmid used togenerate the AAV406 vector.

SEQ ID NO: 109 sets forth the nucleotide sequence of a plasmid used togenerate the AAV-CAR100 (AAV408) vector.

SEQ ID NO: 110 sets forth the nucleotide sequence of a plasmid used togenerate the AAV-CAR763 (AAV412) vector.

SEQ ID NO: 111 sets forth the amino acid sequence of an anti-CD19chimeric antigen receptor.

SEQ ID NO: 112 sets forth the amino acid sequence of an anti-CD19extracellular ligand-binding domain.

SEQ ID NO: 113 sets forth the amino acid sequence of a chimeric antigenreceptor intracellular cytoplasmic signaling domain.

SEQ ID NO: 114 sets forth the amino acid sequence of a chimeric antigenreceptor intracellular co-stimulatory domain.

SEQ ID NO: 115 sets forth the amino acid sequence of a chimeric antigenreceptor signal peptide domain.

SEQ ID NO: 116 sets forth the amino acid sequence of a chimeric antigenreceptor hinge region.

SEQ ID NO: 117 sets forth the amino acid sequence of a chimeric antigenreceptor transmembrane domain.

SEQ ID NO: 118 sets forth the nucleotide sequence of an EF-1 alpha corepromoter.

SEQ ID NO: 119 sets forth the nucleotide sequence of an exogenouspolynucleotide insert.

SEQ ID NO: 120 sets forth the nucleotide sequence of the human TCR alphaconstant region gene comprising an exogenous nucleic acid sequenceinserted within the TRC 1-2 recognition sequence.

SEQ ID NO: 121 sets forth the nucleotide sequence of the human TCR alphaconstant region gene comprising an exogenous nucleic acid sequenceinserted within the TRC 3-4 recognition sequence.

SEQ ID NO: 122 sets forth the nucleotide sequence of the human TCR alphaconstant region gene comprising an exogenous nucleic acid sequenceinserted within the TRC 7-8 recognition sequence.

SEQ ID NO: 123 sets forth the nucleic acid sequence of a plasmid used togenerate the AAV421 vector.

SEQ ID NO: 124 sets forth the nucleic acid sequence of a plasmid used togenerate the AAV422 vector.

SEQ ID NO: 125 sets forth the nucleic acid sequence of a plasmid used togenerate the AAV423 vector.

DETAILED DESCRIPTION OF THE INVENTION 1.1 References and Definitions

The patent and scientific literature referred to herein establishesknowledge that is available to those of skill in the art. The issued USpatents, allowed applications, published foreign applications, andreferences, including GenBank database sequences, which are cited hereinare hereby incorporated by reference to the same extent as if each wasspecifically and individually indicated to be incorporated by reference.

The present invention can be embodied in different forms and should notbe construed as limited to the embodiments set forth herein. Rather,these embodiments are provided so that this disclosure will be thoroughand complete, and will fully convey the scope of the invention to thoseskilled in the art. For example, features illustrated with respect toone embodiment can be incorporated into other embodiments, and featuresillustrated with respect to a particular embodiment can be deleted fromthat embodiment. In addition, numerous variations and additions to theembodiments suggested herein will be apparent to those skilled in theart in light of the instant disclosure, which do not depart from theinstant invention.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The terminology used in thedescription of the invention herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference herein in their entirety.

As used herein, “a,” “an,” or “the” can mean one or more than one. Forexample, “a” cell can mean a single cell or a multiplicity of cells.

As used herein, unless specifically indicated otherwise, the word “or”is used in the inclusive sense of “and/or” and not the exclusive senseof “either/or.”

As used herein, the term “meganuclease” refers to an endonuclease thatbinds double-stranded DNA at a recognition sequence that is greater than12 base pairs. Preferably, the recognition sequence for a meganucleaseof the invention is 22 base pairs. A meganuclease can be an endonucleasethat is derived from I-CreI, and can refer to an engineered variant ofI-CreI that has been modified relative to natural I-CreI with respectto, for example, DNA-binding specificity, DNA cleavage activity,DNA-binding affinity, or dimerization properties. Methods for producingsuch modified variants of I-CreI are known in the art (e.g. WO2007/047859). A meganuclease as used herein binds to double-stranded DNAas a heterodimer or as a “single-chain meganuclease” in which a pair ofDNA-binding domains are joined into a single polypeptide using a peptidelinker. The term “homing endonuclease” is synonymous with the term“meganuclease.” Meganucleases of the invention are substantiallynon-toxic when expressed in cells, particularly in human T cells, suchthat cells can be transfected and maintained at 37° C. without observingdeleterious effects on cell viability or significant reductions inmeganuclease cleavage activity when measured using the methods describedherein.

As used herein, the term “single-chain meganuclease” refers to apolypeptide comprising a pair of meganuclease subunits joined by alinker. A single-chain meganuclease has the organization: N-terminalsubunit—Linker—C-terminal subunit. The two meganuclease subunits willgenerally be non-identical in amino acid sequence and will recognizenon-identical DNA sequences. Thus, single-chain meganucleases typicallycleave pseudo-palindromic or non-palindromic recognition sequences. Asingle-chain meganuclease may be referred to as a “single-chainheterodimer” or “single-chain heterodimeric meganuclease” although it isnot, in fact, dimeric. For clarity, unless otherwise specified, the term“meganuclease” can refer to a dimeric or single-chain meganuclease.

As used herein, the term “linker” refers to an exogenous peptidesequence used to join two meganuclease subunits into a singlepolypeptide. A linker may have a sequence that is found in naturalproteins, or may be an artificial sequence that is not found in anynatural protein. A linker may be flexible and lacking in secondarystructure or may have a propensity to form a specific three-dimensionalstructure under physiological conditions. A linker can include, withoutlimitation, those encompassed by U.S. Pat. No. 8,445,251. In someembodiments, a linker may have an amino acid sequence comprisingresidues 154-195 of any one of SEQ ID NOs:8-32.

As used herein, with respect to a protein, the term “recombinant” meanshaving an altered amino acid sequence as a result of the application ofgenetic engineering techniques to nucleic acids that encode the protein,and cells or organisms that express the protein. With respect to anucleic acid, the term “recombinant” means having an altered nucleicacid sequence as a result of the application of genetic engineeringtechniques. Genetic engineering techniques include, but are not limitedto, PCR and DNA cloning technologies; transfection, transformation andother gene transfer technologies; homologous recombination;site-directed mutagenesis; and gene fusion. In accordance with thisdefinition, a protein having an amino acid sequence identical to anaturally-occurring protein, but produced by cloning and expression in aheterologous host, is not considered recombinant.

As used herein, the term “wild-type” refers to the most common naturallyoccurring allele (i.e., polynucleotide sequence) in the allelepopulation of the same type of gene, wherein a polypeptide encoded bythe wild-type allele has its original functions. The term “wild-type”also refers a polypeptide encoded by a wild-type allele. Wild-typealleles (i.e., polynucleotides) and polypeptides are distinguishablefrom mutant or variant alleles and polypeptides, which comprise one ormore mutations and/or substitutions relative to the wild-typesequence(s). Whereas a wild-type allele or polypeptide can confer anormal phenotype in an organism, a mutant or variant allele orpolypeptide can, in some instances, confer an altered phenotype.Wild-type homing endonucleases are distinguishable from recombinant ornon-naturally-occurring meganucleases.

As used herein with respect to recombinant proteins, the term“modification” means any insertion, deletion or substitution of an aminoacid residue in the recombinant sequence relative to a referencesequence (e.g., a wild-type or a native sequence).

As used herein, the term “recognition sequence” refers to a DNA sequencethat is bound and cleaved by a meganuclease. In the case of arecombinant meganuclease of the invention, a recognition sequencecomprises a pair of inverted, 9 base pair “half-sites” or “recognitionhalf-sites” that are separated by four base pairs. In the case of asingle-chain meganuclease, the N-terminal subunit of the proteincontacts a first half-site and the C-terminal subunit of the proteincontacts a second half-site. Cleavage by a meganuclease produces fourbase pair 3′ “overhangs”. “Overhangs”, or “sticky ends” are short,single-stranded DNA segments that can be produced by meganucleasecleavage of a double-stranded DNA sequence. In the case of meganucleasesof the present invention, the overhang comprises bases 10-13 of the 22base pair recognition sequence.

As used herein, the term “target site” or “target sequence” refers to aregion of the chromosomal DNA of a cell comprising a recognitionsequence for a meganuclease.

As used herein, the term “DNA-binding affinity” or “binding affinity”means the tendency of a meganuclease to non-covalently associate with areference DNA molecule (e.g., a recognition sequence or an arbitrarysequence). Binding affinity is measured by a dissociation constant,K_(d). As used herein, a meganuclease has “altered” binding affinity ifthe K_(d) of the recombinant meganuclease for a reference recognitionsequence is increased or decreased by a statistically significantpercent change relative to a reference meganuclease.

As used herein, the term “homologous recombination” or “HR” refers tothe natural, cellular process in which a double-stranded DNA-break isrepaired using a homologous DNA sequence as the repair template (see,e.g., Cahill et al. (2006), Front. Biosci. 11:1958-1976). The homologousDNA sequence may be an endogenous chromosomal sequence or an exogenousnucleic acid that was delivered to the cell.

As used herein, the term “non-homologous end-joining” or “NHEJ” refersto the natural, cellular process in which a double-stranded DNA-break isrepaired by the direct joining of two non-homologous DNA segments (see,e.g., Cahill et al. (2006), Front. Biosci. 11:1958-1976). DNA repair bynon-homologous end-joining is error-prone and frequently results in theuntemplated addition or deletion of DNA sequences at the site of repair.In some instances, cleavage at a target recognition sequence results inNHEJ at a target recognition site. Nuclease-induced cleavage of a targetsite in the coding sequence of a gene followed by DNA repair by NHEJ canintroduce mutations into the coding sequence, such as frameshiftmutations, that disrupt gene function. Thus, engineered nucleases suchas meganucleases can be used to effectively knock-out a gene in apopulation of cells.

As used herein, a “chimeric antigen receptor” or “CAR” refers to anengineered receptor that confers or grafts specificity for an antigenonto an immune effector cell (e.g., a human T cell). A chimeric antigenreceptor typically comprises an extracellular ligand-binding domain ormoiety and an intracellular domain that comprises one or morestimulatory domains that transduce the signals necessary for T cellactivation. In some embodiments, the extracellular ligand-binding domainor moiety can be in the form of single-chain variable fragments (scFvs)derived from a monoclonal antibody, which provide specificity for aparticular epitope or antigen (e.g., an epitope or antigenpreferentially present on the surface of a cancer cell or otherdisease-causing cell or particle). The extracellular ligand-bindingdomain can be specific for any antigen or epitope of interest. In aparticular embodiment, the ligand-binding domain is specific for CD19.In other embodiments, the scFv can be humanized.

The extracellular domain of a chimeric antigen receptor can alsocomprise an autoantigen (see, Payne et al. (2016), Science353(6295):179-184), which can be recognized by autoantigen-specific Bcell receptors on B lymphocytes, thus directing T cells to specificallytarget and kill autoreactive B lymphocytes in antibody-mediatedautoimmune diseases. Such CARs can be referred to as chimericautoantibody receptors (CAARs), and their use is encompassed by theinvention.

The scFvs can be attached via a linker sequence. The intracellularstimulatory domain can include one or more cytoplasmic signaling domainsthat transmit an activation signal to the immune effector cell followingantigen binding. Such cytoplasmic signaling domains can include, withoutlimitation, CD3-zeta. The intracellular stimulatory domain can alsoinclude one or more intracellular co-stimulatory domains that transmit aproliferative and/or cell-survival signal after ligand binding. Suchintracellular co-stimulatory domains can include, without limitation, aCD28 domain, a 4-1BB domain, an OX40 domain, or a combination thereof. Achimeric antigen receptor can further include additional structuralelements, including a transmembrane domain that is attached to theextracellular ligand-binding domain via a hinge or spacer sequence.

As used herein, an “exogenous T cell receptor” or “exogenous TCR” refersto a TCR whose sequence is introduced into the genome of an immuneeffector cell (e.g., a human T cell) that may or may not endogenouslyexpress the TCR. Expression of an exogenous TCR on an immune effectorcell can confer specificity for a specific epitope or antigen (e.g., anepitope or antigen preferentially present on the surface of a cancercell or other disease-causing cell or particle). Such exogenous T cellreceptors can comprise alpha and beta chains or, alternatively, maycomprise gamma and delta chains. Exogenous TCRs useful in the inventionmay have specificity to any antigen or epitope of interest.

As used herein, the term “reduced expression” refers to any reduction inthe expression of the endogenous T cell receptor at the cell surface ofa genetically-modified cell when compared to a control cell. The termreduced can also refer to a reduction in the percentage of cells in apopulation of cells that express an endogenous polypeptide (i.e., anendogenous T cell receptor) at the cell surface when compared to apopulation of control cells. Such a reduction may be up to 5%, 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or up to 100%. Accordingly, theterm “reduced” encompasses both a partial knockdown and a completeknockdown of the endogenous T cell receptor.

As used herein with respect to both amino acid sequences and nucleicacid sequences, the terms “percent identity,” “sequence identity,”“percentage similarity,” “sequence similarity” and the like refer to ameasure of the degree of similarity of two sequences based upon analignment of the sequences that maximizes similarity between alignedamino acid residues or nucleotides, and that is a function of the numberof identical or similar residues or nucleotides, the number of totalresidues or nucleotides, and the presence and length of gaps in thesequence alignment. A variety of algorithms and computer programs areavailable for determining sequence similarity using standard parameters.As used herein, sequence similarity is measured using the BLASTp programfor amino acid sequences and the BLASTn program for nucleic acidsequences, both of which are available through the National Center forBiotechnology Information website, and are described in, for example,Altschul et al. (1990), J. Mol. Biol. 215:403-410; Gish and States(1993), Nature Genet. 3:266-272; Madden et al. (1996), Meth. Enzymol.266:131-141; Altschul et al. (1997), Nucleic Acids Res. 25:33 89-3402;Zhang et al. (2000), J. Comput. Biol. 7(1-2):203-14. As used herein,percent similarity of two amino acid sequences is the score based uponthe following parameters for the BLASTp algorithm: word size=3; gapopening penalty=−11; gap extension penalty=−1; and scoringmatrix=BLOSUM62. As used herein, percent similarity of two nucleic acidsequences is the score based upon the following parameters for theBLASTn algorithm: word size=11; gap opening penalty=−5; gap extensionpenalty=2; match reward=1; and mismatch penalty=−3.

As used herein with respect to modifications of two proteins or aminoacid sequences, the term “corresponding to” is used to indicate that aspecified modification in the first protein is a substitution of thesame amino acid residue as in the modification in the second protein,and that the amino acid position of the modification in the firstproteins corresponds to or aligns with the amino acid position of themodification in the second protein when the two proteins are subjectedto standard sequence alignments (e.g., using the BLASTp program). Thus,the modification of residue “X” to amino acid “A” in the first proteinwill correspond to the modification of residue “Y” to amino acid “A” inthe second protein if residues X and Y correspond to each other in asequence alignment, and despite the fact that X and Y may be differentnumbers.

As used herein, the term “recognition half-site,” “recognition sequencehalf-site,” or simply “half-site” means a nucleic acid sequence in adouble-stranded DNA molecule that is recognized by a monomer of ahomodimeric or heterodimeric meganuclease, or by one subunit of asingle-chain meganuclease.

As used herein, the term “hypervariable region” refers to a localizedsequence within a meganuclease monomer or subunit that comprises aminoacids with relatively high variability. A hypervariable region cancomprise about 50-60 contiguous residues, about 53-57 contiguousresidues, or preferably about 56 residues. In some embodiments, theresidues of a hypervariable region may correspond to positions 24-79 orpositions 215-270 of any one of SEQ ID NOs:8-32. A hypervariable regioncan comprise one or more residues that contact DNA bases in arecognition sequence and can be modified to alter base preference of themonomer or subunit. A hypervariable region can also comprise one or moreresidues that bind to the DNA backbone when the meganuclease associateswith a double-stranded DNA recognition sequence. Such residues can bemodified to alter the binding affinity of the meganuclease for the DNAbackbone and the target recognition sequence. In different embodimentsof the invention, a hypervariable region may comprise between 1-20residues that exhibit variability and can be modified to influence basepreference and/or DNA-binding affinity. In particular embodiments, ahypervariable region comprises between about 15-18 residues that exhibitvariability and can be modified to influence base preference and/orDNA-binding affinity. In some embodiments, variable residues within ahypervariable region correspond to one or more of positions 24, 26, 28,29, 30, 32, 33, 38, 40, 42, 44, 46, 66, 68, 70, 72, 73, 75, and 77 ofany one of SEQ ID NOs:8-32. In other embodiments, variable residueswithin a hypervariable region correspond to one or more of positions215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 248, 257, 259,261, 263, 264, 266, and 268 of any one of SEQ ID NOs:8-32.

As used herein, the terms “T cell receptor alpha constant region gene”and “TCR alpha constant region gene” are used interchangeably and referto the human gene identified by NCBI Gen ID NO. 28755 (SEQ ID NO:1).

The terms “recombinant DNA construct,” “recombinant construct,”“expression cassette,” “expression construct,” “chimeric construct,”“construct,” and “recombinant DNA fragment” are used interchangeablyherein and are single or double-stranded polynucleotides. A recombinantconstruct comprises an artificial combination of single ordouble-stranded polynucleotides, including, without limitation,regulatory and coding sequences that are not found together in nature.For example, a recombinant DNA construct may comprise regulatorysequences and coding sequences that are derived from different sources,or regulatory sequences and coding sequences derived from the samesource and arranged in a manner different than that found in nature.Such a construct may be used by itself or may be used in conjunctionwith a vector.

As used herein, a “vector” or “recombinant DNA vector” may be aconstruct that includes a replication system and sequences that arecapable of transcription and translation of a polypeptide-encodingsequence in a given host cell. If a vector is used then the choice ofvector is dependent upon the method that will be used to transform hostcells as is well known to those skilled in the art. Vectors can include,without limitation, plasmid vectors and recombinant AAV vectors, or anyother vector known in that art suitable for delivering a gene encoding ameganuclease of the invention to a target cell. The skilled artisan iswell aware of the genetic elements that must be present on the vector inorder to successfully transform, select and propagate host cellscomprising any of the isolated nucleotides or nucleic acid sequences ofthe invention.

As used herein, a “vector” can also refer to a viral vector. Viralvectors can include, without limitation, retroviral vectors, lentiviralvectors, adenoviral vectors, and adeno-associated viral vectors (AAV).

As used herein, a “polycistronic” mRNA refers to a single messenger RNAthat comprises two or more coding sequences (i.e., cistrons) and encodesmore than one protein. A polycistronic mRNA can comprise any elementknown in the art to allow for the translation of two or more genes fromthe same mRNA molecule including, but not limited to, an IRES element, aT2A element, a P2A element, an E2A element, and an F2A element.

As used herein, a “human T cell” or “T cell” refers to a T cell isolatedfrom a human donor. Human T cells, and cells derived therefrom, includeisolated T cells that have not been passaged in culture, T cells thathave been passaged and maintained under cell culture conditions withoutimmortalization, and T cells that have been immortalized and can bemaintained under cell culture conditions indefinitely.

As used herein, a “control” or “control cell” refers to a cell thatprovides a reference point for measuring changes in genotype orphenotype of a genetically-modified cell. A control cell may comprise,for example: (a) a wild-type cell, i.e., of the same genotype as thestarting material for the genetic alteration that resulted in thegenetically-modified cell; (b) a cell of the same genotype as thegenetically-modified cell but that has been transformed with a nullconstruct (i.e., with a construct that has no known effect on the traitof interest); or, (c) a cell genetically identical to thegenetically-modified cell but that is not exposed to conditions orstimuli or further genetic modifications that would induce expression ofaltered genotype or phenotype.

As used herein, the recitation of a numerical range for a variable isintended to convey that the invention may be practiced with the variableequal to any of the values within that range. Thus, for a variable thatis inherently discrete, the variable can be equal to any integer valuewithin the numerical range, including the end-points of the range.Similarly, for a variable that is inherently continuous, the variablecan be equal to any real value within the numerical range, including theend-points of the range. As an example, and without limitation, avariable that is described as having values between 0 and 2 can take thevalues 0, 1 or 2 if the variable is inherently discrete, and can takethe values 0.0, 0.1, 0.01, 0.001, or any other real values ≥0 and ≤2 ifthe variable is inherently continuous.

2.1 Principle of the Invention

The present invention is based, in part, on the discovery thatrecombinant meganucleases can be engineered to recognize and cleaverecognition sequences found within the human TCR alpha constant regiongene (SEQ ID NO:1), particularly within residues 93-208. Cleavage atsuch recognition sequences can allow for NHEJ at the cleavage site anddisrupted expression of the human T cell receptor alpha chain subunit,leading to reduced expression and/or function of the T cell receptor atthe cell surface.

Additionally, cleavage at such recognition sequences can further allowfor homologous recombination of exogenous nucleic acid sequencesdirectly into the TCR alpha constant region gene. Such exogenous nucleicacid sequences can comprise a sequence of interest, such as a sequenceencoding a chimeric antigen receptor, an exogenous TCR receptor, or anyother polypeptide of interest. Thus, the present invention allows forboth the knockout of the endogenous T cell receptor and the expressionof an exogenous nucleic acid sequence (e.g., a chimeric antigen receptoror exogenous TCR) by targeting a single recognition site with a singleengineered nuclease. Such cells can exhibit reduced or no induction ofgraft-versus-host-disease (GVHD) when administered to an allogeneicsubject.

2.2 Meganucleases for Recognizing and Cleaving Recognition SequencesWithin the T Cell Receptor Alpha Constant Region Gene

It is known in the art that it is possible to use a site-specificnuclease to make a DNA break in the genome of a living cell, and thatsuch a DNA break can result in permanent modification of the genome viamutagenic NHEJ repair or via homologous recombination with a transgenicDNA sequence. NHEJ can produce mutagenesis at the cleavage site,resulting in inactivation of the allele. NHEJ-associated mutagenesis mayinactivate an allele via generation of early stop codons, frameshiftmutations producing aberrant non-functional proteins, or could triggermechanisms such as nonsense-mediated mRNA decay. The use ofmeganucleases to induce mutagenesis via NHEJ can be used to target aspecific mutation or a sequence present in a wild-type allele. The useof meganucleases to induce a double-strand break in a target locus isknown to stimulate homologous recombination, particularly of transgenicDNA sequences flanked by sequences that are homologous to the genomictarget. In this manner, exogenous nucleic acid sequences can be insertedinto a target locus. Such exogenous nucleic acids can encode, forexample, a chimeric antigen receptor, an exogenous TCR, or any sequenceor polypeptide of interest.

In preferred embodiments, the nucleases used to practice the inventionare single-chain meganucleases. A single-chain meganuclease comprises anN-terminal subunit and a C-terminal subunit joined by a linker peptide.Each of the two domains recognizes half of the recognition sequence(i.e., a recognition half-site) and the site of DNA cleavage is at themiddle of the recognition sequence near the interface of the twosubunits. DNA strand breaks are offset by four base pairs such that DNAcleavage by a meganuclease generates a pair of four base pair, 3′single-strand overhangs.

In some examples, recombinant meganucleases of the invention have beenengineered to recognize and cleave the TRC 1-2 recognition sequence (SEQID NO:3). Such recombinant meganucleases are collectively referred toherein as “TRC 1-2 meganucleases.” Exemplary TRC 1-2 meganucleases areprovided in SEQ ID NOs:8-27.

In additional examples, recombinant meganucleases of the invention havebeen engineered to recognize and cleave the TRC 3-4 recognition sequence(SEQ ID NO:4). Such recombinant meganucleases are collectively referredto herein as “TRC 3-4 meganucleases.” Exemplary TRC 3-4 meganucleasesare provided in SEQ ID NOs:28 and 29.

In further examples, recombinant meganucleases of the invention havebeen engineered to recognize and cleave the TRC 7-8 recognition sequence(SEQ ID NO:5). Such recombinant meganucleases are collectively referredto herein as “TRC 7-8 meganucleases.” Exemplary TRC 7-8 meganucleasesare provided in SEQ ID NOs:30-32.

Recombinant meganucleases of the invention comprise a first subunit,comprising a first hypervariable (HVR1) region, and a second subunit,comprising a second hypervariable (HVR2) region. Further, the firstsubunit binds to a first recognition half-site in the recognitionsequence (e.g., the TRC1, TRC3, or TRC7 half-site), and the secondsubunit binds to a second recognition half-site in the recognitionsequence (e.g., the TRC2, TRC4, or TRC8 half-site). In embodiments wherethe recombinant meganuclease is a single-chain meganuclease, the firstand second subunits can be oriented such that the first subunit, whichcomprises the HVR1 region and binds the first half-site, is positionedas the N-terminal subunit, and the second subunit, which comprises theHVR2 region and binds the second half-site, is positioned as theC-terminal subunit. In alternative embodiments, the first and secondsubunits can be oriented such that the first subunit, which comprisesthe HVR1 region and binds the first half-site, is positioned as theC-terminal subunit, and the second subunit, which comprises the HVR2region and binds the second half-site, is positioned as the N-terminalsubunit. Exemplary TRC 1-2 meganucleases of the invention are providedin Table 1. Exemplary TRC 3-4 meganucleases of the invention areprovided in Table 2. Exemplary TRC 7-8 meganucleases of the inventionare provided in Table 3.

TABLE 1 Exemplary recombinant meganucleases engineered to recognize andcleave the TRC 1-2 recognition sequence (SEQ ID NO: 3) AA TRC1 TRC1*TRC1 TRC2 TRC2 *TRC2 SEQ Subunit Subunit Subunit Subunit SubunitSubunit Meganuclease ID Residues SEQ ID % Residues SEQ ID % TRC 1-2x.87EE 8 198-344 33 100  7-153 58 100 TRC 1-2x.87 QE 9 198-344 34 100  7-15359 99.3 TRC 1-2x.87 EQ 10 198-344 35 99.3  7-153 60 100 TRC 1-2x.87 11198-344 36 99.3  7-153 61 99.3 TRC 1-2x.6 12 198-344 37 99.3  7-153 6294.6 TRC 1-2x.20 13 198-344 38 99.3  7-153 63 91.2 TRC 1-2x.55 14198-344 39 95.9  7-153 64 91.8 TRC 1-2x.60 15 198-344 40 91.8  7-153 6591.2 TRC 1-2x.105 16 198-344 41 95.2  7-153 66 95.2 TRC 1-2x.163 17198-344 42 99.3  7-153 67 99.3 TRC 1-2x.113_3 18 198-344 43 99.3  7-15368 91.2 TRC 1-2x.5 19  7-153 44 99.3 198-344 69 93.2 TRC 1-2x.8 20 7-153 45 92.5 198-344 70 92.5 TRC 1-2x.25 21  7-153 46 99.3 198-344 7198.6 TRC 1-2x.72 22  7-153 47 99.3 198-344 72 92.5 TRC 1-2x.80 23  7-15348 99.3 198-344 73 92.5 TRC 1-2x.84 24  7-153 49 95.2 198-344 74 98.6TRC 1-2x.120 25  7-153 50 99.3 198-344 75 92.5 TRC 1-2x.113_1 26  7-15351 100 198-344 76 92.5 TRC 1-2x.113_2 27  7-153 52 99.3 198-344 77 92.5*“TRC1 Subunit %” and “TRC2 Subunit %” represent the amino acid sequenceidentity between the TRC1-binding and TRC2-binding subunit regions ofeach meganuclease and the TRC1-binding and TRC2-binding subunit regions,respectively, of the TRC 1-2x.87 EE meganuclease.

TABLE 2 Exemplary recombinant meganucleases engineered to recognize andcleave the TRC 3-4 recognition sequence (SEQ ID NO: 4) AA TRC3 TRC3*TRC3 TRC4 TRC4 *TRC4 SEQ Subunit Subunit Subunit Subunit SubunitSubunit Meganuclease ID Residues SEQ ID % Residues SEQ ID % TRC 3-4x.328 7-153 53 100 198-344 78 100 TRC 3-4x.19 29 7-153 54 96.6 198-344 7996.6 *“TRC3 Subunit %” and “TRC4 Subunit %” represent the amino acidsequence identity between the TRC3-binding and TRC4-binding subunitregions of each meganuclease and the TRC3-binding and TRC4-bindingsubunit regions, respectively, of the TRC 3-4x.3 meganuclease.

TABLE 3 Exemplary recombinant meganucleases engineered to recognize andcleave the TRC 7-8 recognition sequence (SEQ ID NO: 5) AA TRC7 TRC7*TRC7 TRC8 TRC8 *TRC8 SEQ Subunit Subunit Subunit Subunit SubunitSubunit Meganuclease ID Residues SEQ ID % Residues SEQ ID % TRC 7-8x.730  7-153 55 100 198-344  80 100 TRC 7-8x.9 31 198-344 56 97.3 7-153 8191.2 TRC 7-8x.14 32 198-344 57 97.9 7-153 82 90.5 *“TRC7 Subunit %” and“TRC8 Subunit %” represent the amino acid sequence identity between theTRC7-binding and TRC8-binding subunit regions of each meganuclease andthe TRC7-binding and TRC8-binding subunit regions, respectively, of theTRC 7-8x.7 meganuclease.

2.3 Methods for Producing Genetically-Modified Cells

The invention provides methods for producing genetically-modified cellsusing recombinant meganucleases that recognize and cleave recognitionsequences found within the human TCR alpha constant region gene (SEQ IDNO:1). Cleavage at such recognition sequences can allow for NHEJ at thecleavage site and disrupted expression of the human T cell receptoralpha chain subunit, leading to reduced expression and/or function ofthe T cell receptor at the cell surface. Additionally, cleavage at suchrecognition sequences can further allow for homologous recombination ofexogenous nucleic acid sequences directly into the TCR alpha constantregion gene.

Recombinant meganucleases of the invention can be delivered into a cellin the form of protein or, preferably, as a nucleic acid encoding therecombinant meganuclease. Such nucleic acid can be DNA (e.g., circularor linearized plasmid DNA or PCR products) or RNA.

For embodiments in which the recombinant meganuclease coding sequence isdelivered in DNA form, it should be operably linked to a promoter tofacilitate transcription of the meganuclease gene. Mammalian promoterssuitable for the invention include constitutive promoters such as thecytomegalovirus early (CMV) promoter (Thomsen et al. (1984), Proc NatlAcad Sci USA. 81(3):659-63) or the SV40 early promoter (Benoist andChambon (1981), Nature. 290(5804):304-10) as well as inducible promoterssuch as the tetracycline-inducible promoter (Dingermann et al. (1992),Mol Cell Biol. 12(9):4038-45).

In some embodiments, mRNA encoding the engineered nuclease is deliveredto the cell because this reduces the likelihood that the gene encodingthe engineered nuclease will integrate into the genome of the cell. SuchmRNA encoding an engineered nuclease can be produced using methods knownin the art such as in vitro transcription. In some embodiments, the mRNAis capped using 7-methyl-guanosine. In some embodiments, the mRNA may bepolyadenylated.

In particular embodiments, an mRNA encoding an engineered nuclease ofthe invention can be a polycistronic mRNA encoding two or more nucleasesthat are simultaneously expressed in the cell. A polycistronic mRNA canencode two or more nucleases of the invention that target differentrecognition sequences in the same target gene. Alternatively, apolycistronic mRNA can encode at least one nuclease described herein andat least one additional nuclease targeting a separate recognitionsequence positioned in the same gene, or targeting a second recognitionsequence positioned in a second gene such that cleavage sites areproduced in both genes. A polycistronic mRNA can comprise any elementknown in the art to allow for the translation of two or more genes(i.e., cistrons) from the same mRNA molecule including, but not limitedto, an IRES element, a T2A element, a P2A element, an E2A element, andan F2A element.

Purified recombinant meganuclease proteins can be delivered into cellsto cleave genomic DNA, which allows for homologous recombination ornon-homologous end-joining at the cleavage site with a sequence ofinterest, by a variety of different mechanisms known in the art.

In some embodiments, recombinant meganuclease proteins, or DNA/mRNAencoding recombinant meganucleases, are coupled to a cell penetratingpeptide or targeting ligand to facilitate cellular uptake. Examples ofcell penetrating peptides known in the art include poly-arginine(Jearawiriyapaisarn, et al. (2008) Mol Ther. 16:1624-9), TAT peptidefrom the HIV virus (Hudecz et al. (2005), Med. Res. Rev. 25: 679-736),MPG (Simeoni, et al. (2003) Nucleic Acids Res. 31:2717-2724), Pep-1(Deshayes et al. (2004) Biochemistry 43: 7698-7706, and HSV-1 VP-22(Deshayes et al. (2005) Cell Mol Life Sci. 62:1839-49. In an alternativeembodiment, recombinant meganucleases, or DNA/mRNA encoding recombinantmeganucleases, are coupled covalently or non-covalently to an antibodythat recognizes a specific cell-surface receptor expressed on targetcells such that the meganuclease protein/DNA/mRNA binds to and isinternalized by the target cells. Alternatively, recombinantmeganuclease protein/DNA/mRNA can be coupled covalently ornon-covalently to the natural ligand (or a portion of the naturalligand) for such a cell-surface receptor. (McCall, et al. (2014) TissueBarriers. 2(4):e944449; Dinda, et al. (2013) Curr Pharm Biotechnol.14:1264-74; Kang, et al. (2014) Curr Pharm Biotechnol. 15(3):220-30;Qian et al. (2014) Expert Opin Drug Metab Toxicol. 10(11):1491-508).

In some embodiments, recombinant meganuclease proteins, or DNA/mRNAencoding recombinant meganucleases, are coupled covalently or,preferably, non-covalently to a nanoparticle or encapsulated within sucha nanoparticle using methods known in the art (Sharma, et al. (2014)Biomed Res Int. 2014). A nanoparticle is a nanoscale delivery systemwhose length scale is <1 μm, preferably <100 nm. Such nanoparticles maybe designed using a core composed of metal, lipid, polymer, orbiological macromolecule, and multiple copies of the recombinantmeganuclease proteins, mRNA, or DNA can be attached to or encapsulatedwith the nanoparticle core. This increases the copy number of theprotein/mRNA/DNA that is delivered to each cell and, so, increases theintracellular expression of each recombinant meganuclease to maximizethe likelihood that the target recognition sequences will be cut. Thesurface of such nanoparticles may be further modified with polymers orlipids (e.g., chitosan, cationic polymers, or cationic lipids) to form acore-shell nanoparticle whose surface confers additional functionalitiesto enhance cellular delivery and uptake of the payload (Jian et al.(2012) Biomaterials. 33(30): 7621-30). Nanoparticles may additionally beadvantageously coupled to targeting molecules to direct the nanoparticleto the appropriate cell type and/or increase the likelihood of cellularuptake. Examples of such targeting molecules include antibodies specificfor cell-surface receptors and the natural ligands (or portions of thenatural ligands) for cell surface receptors.

In some embodiments, the meganuclease proteins or DNA/mRNA encoding themeganucleases are encapsulated within liposomes or complexed usingcationic lipids (see, e.g., Lipofectamine™, Life Technologies Corp.,Carlsbad, Calif.; Zuris et al. (2015) Nat Biotechnol. 33: 73-80; Mishraet al. (2011) J Drug Deliv. 2011:863734). The liposome and lipoplexformulations can protect the payload from degradation, and facilitatecellular uptake and delivery efficiency through fusion with and/ordisruption of the cellular membranes of the cells.

In some embodiments, recombinant meganuclease proteins, or DNA/mRNAencoding recombinant meganucleases, are encapsulated within polymericscaffolds (e.g., PLGA) or complexed using cationic polymers (e.g., PEI,PLL) (Tamboli et al. (2011) Ther Deliv. 2(4): 523-536).

In some embodiments, recombinant meganuclease proteins, or DNA/mRNAencoding recombinant meganucleases, are combined with amphiphilicmolecules that self-assemble into micelles (Tong et al. (2007) J GeneMed. 9(11): 956-66). Polymeric micelles may include a micellar shellformed with a hydrophilic polymer (e.g., polyethyleneglycol) that canprevent aggregation, mask charge interactions, and reduce nonspecificinteractions outside of the cell.

In some embodiments, recombinant meganuclease proteins, or DNA/mRNAencoding recombinant meganucleases, are formulated into an emulsion or ananoemulsion (i.e., having an average particle diameter of <1 nm) fordelivery to the cell. The term “emulsion” refers to, without limitation,any oil-in-water, water-in-oil, water-in-oil-in-water, oroil-in-water-in-oil dispersions or droplets, including lipid structuresthat can form as a result of hydrophobic forces that drive apolarresidues (e.g., long hydrocarbon chains) away from water and polar headgroups toward water, when a water immiscible phase is mixed with anaqueous phase. These other lipid structures include, but are not limitedto, unilamellar, paucilamellar, and multilamellar lipid vesicles,micelles, and lamellar phases. Emulsions are composed of an aqueousphase and a lipophilic phase (typically containing an oil and an organicsolvent). Emulsions also frequently contain one or more surfactants.Nanoemulsion formulations are well known, e.g., as described in USPatent Application Nos. 2002/0045667 and 2004/0043041, and U.S. Pat.Nos. 6,015,832, 6,506,803, 6,635,676, and 6,559,189, each of which isincorporated herein by reference in its entirety.

In some embodiments, recombinant meganuclease proteins, or DNA/mRNAencoding recombinant meganucleases, are covalently attached to, ornon-covalently associated with, multifunctional polymer conjugates, DNAdendrimers, and polymeric dendrimers (Mastorakos et al. (2015)Nanoscale. 7(9): 3845-56; Cheng et al. (2008) J Pharm Sci. 97(1):123-43). The dendrimer generation can control the payload capacity andsize, and can provide a high payload capacity. Moreover, display ofmultiple surface groups can be leveraged to improve stability and reducenonspecific interactions.

In some embodiments, genes encoding a recombinant meganuclease areintroduced into a cell using a viral vector. Such vectors are known inthe art and include lentiviral vectors, adenoviral vectors, andadeno-associated virus (AAV) vectors (reviewed in Vannucci, et al. (2013New Microbiol. 36:1-22). Recombinant AAV vectors useful in the inventioncan have any serotype that allows for transduction of the virus into thecell and insertion of the meganuclease gene into the cell genome. Inparticular embodiments, recombinant AAV vectors have a serotype of AAV2or AAV6. Recombinant AAV vectors can also be self-complementary suchthat they do not require second-strand DNA synthesis in the host cell(McCarty, et al. (2001) Gene Ther. 8:1248-54).

If the recombinant meganuclease genes are delivered in DNA form (e.g.plasmid) and/or via a viral vector (e.g. AAV) they must be operablylinked to a promoter. In some embodiments, this can be a viral promotersuch as endogenous promoters from the viral vector (e.g. the LTR of alentiviral vector) or the well-known cytomegalovirus- or SV40virus-early promoters. In a preferred embodiment, meganuclease genes areoperably linked to a promoter that drives gene expression preferentiallyin the target cell (e.g., a human T cell).

The invention further provides for the introduction of an exogenousnucleic acid into the cell, such that the exogenous nucleic acidsequence is inserted into the TRC alpha constant region gene at anuclease cleavage site. In some embodiments, the exogenous nucleic acidcomprises a 5′ homology arm and a 3′ homology arm to promoterecombination of the nucleic acid sequence into the cell genome at thenuclease cleavage site.

Exogenous nucleic acids of the invention may be introduced into the cellby any of the means previously discussed. In a particular embodiment,exogenous nucleic acids are introduced by way of a viral vector, such asa lentivirus, retrovirus, adenovirus, or preferably a recombinant AAVvector. Recombinant AAV vectors useful for introducing an exogenousnucleic acid can have any serotype that allows for transduction of thevirus into the cell and insertion of the exogenous nucleic acid sequenceinto the cell genome. In particular embodiments, the recombinant AAVvectors have a serotype of AAV2 or AAV6. The recombinant AAV vectors canalso be self-complementary such that they do not require second-strandDNA synthesis in the host cell.

In another particular embodiment, an exogenous nucleic acid can beintroduced into the cell using a single-stranded DNA template. Thesingle-stranded DNA can comprise the exogenous nucleic acid and, inpreferred embodiments, can comprise 5′ and 3′ homology arms to promoteinsertion of the nucleic acid sequence into the nuclease cleavage siteby homologous recombination. The single-stranded DNA can furthercomprise a 5′ AAV inverted terminal repeat (ITR) sequence 5′ upstream ofthe 5′ homology arm, and a 3′ AAV ITR sequence 3′ downstream of the 3′homology arm.

In another particular embodiment, genes encoding a recombinantmeganuclease of the invention and/or an exogenous nucleic acid sequenceof the invention can be introduced into the cell by transfection with alinearized DNA template. In some examples, a plasmid DNA encoding arecombinant meganuclease and/or an exogenous nucleic acid sequence canbe digested by one or more restriction enzymes such that the circularplasmid DNA is linearized prior to transfection into the cell.

When delivered to a cell, an exogenous nucleic acid of the invention canbe operably linked to any promoter suitable for expression of theencoded polypeptide in the cell, including those mammalian promoters andinducible promoters previously discussed. An exogenous nucleic acid ofthe invention can also be operably linked to a synthetic promoter.Synthetic promoters can include, without limitation, the JeT promoter(WO 2002/012514).

In examples where the genetically-modified cells of the invention arehuman T cells, or cells derived therefrom, such cells may requireactivation prior to introduction of a meganuclease and/or an exogenousnucleic acid sequence. For example, T cells can be contacted withanti-CD3 and anti-CD28 antibodies that are soluble or conjugated to asupport (i.e., beads) for a period of time sufficient to activate thecells.

Genetically-modified cells of the invention can be further modified toexpress one or more inducible suicide genes, the induction of whichprovokes cell death and allows for selective destruction of the cells invitro or in vivo. In some examples, a suicide gene can encode acytotoxic polypeptide, a polypeptide that has the ability to convert anon-toxic pro-drug into a cytotoxic drug, and/or a polypeptide thatactivates a cytotoxic gene pathway within the cell. That is, a suicidegene is a nucleic acid that encodes a product that causes cell death byitself or in the presence of other compounds. A representative exampleof such a suicide gene is one that encodes thymidine kinase of herpessimplex virus. Additional examples are genes that encode thymidinekinase of varicella zoster virus and the bacterial gene cytosinedeaminase that can convert 5-fluorocytosine to the highly toxic compound5-fluorouracil. Suicide genes also include as non-limiting examplesgenes that encode caspase-9, caspase-8, or cytosine deaminase. In someexamples, caspase-9 can be activated using a specific chemical inducerof dimerization (CID). A suicide gene can also encode a polypeptide thatis expressed at the surface of the cell that makes the cells sensitiveto therapeutic and/or cytotoxic monoclonal antibodies. In furtherexamples, a suicide gene can encode recombinant antigenic polypeptidecomprising an antigenic motif recognized by the anti-CD20 mAb Rituximaband an epitope that allows for selection of cells expressing the suicidegene. See, for example, the RQR8 polypeptide described in WO2013153391,which comprises two Rituximab-binding epitopes and a QBEnd10-bindingepitope. For such a gene, Rituximab can be administered to a subject toinduce cell depletion when needed.

2.4 Pharmaceutical Compositions

In some embodiments, the invention provides a pharmaceutical compositioncomprising a genetically-modified cell of the invention, or a populationof genetically-modified cells of the invention, and a pharmaceuticalcarrier. Such pharmaceutical compositions can be prepared in accordancewith known techniques. See, e.g., Remington, The Science And Practice ofPharmacy (21^(st) ed. 2005). In the manufacture of a pharmaceuticalformulation according to the invention, cells are typically admixed witha pharmaceutically acceptable carrier and the resulting composition isadministered to a subject. The carrier must, of course, be acceptable inthe sense of being compatible with any other ingredients in theformulation and must not be deleterious to the subject. In someembodiments, pharmaceutical compositions of the invention can furthercomprise one or more additional agents useful in the treatment of adisease in the subject. In additional embodiments, where thegenetically-modified cell is a genetically-modified human T cell (or acell derived therefrom), pharmaceutical compositions of the inventioncan further include biological molecules, such as cytokines (e.g., IL-2,IL-7, IL-15, and/or IL-21), which promote in vivo cell proliferation andengraftment. Pharmaceutical compositions comprising genetically-modifiedcells of the invention can be administered in the same composition as anadditional agent or biological molecule or, alternatively, can beco-administered in separate compositions.

Pharmaceutical compositions of the invention can be useful for treatingany disease state that can be targeted by T cell adoptive immunotherapy.In a particular embodiment, the pharmaceutical compositions of theinvention are useful in the treatment of cancer. Such cancers caninclude, without limitation, carcinoma, lymphoma, sarcoma, blastomas,leukemia, cancers of B-cell origin, breast cancer, gastric cancer,neuroblastoma, osteosarcoma, lung cancer, melanoma, prostate cancer,colon cancer, renal cell carcinoma, ovarian cancer, rhabdomyo sarcoma,leukemia, and Hodgkin's lymphoma. In certain embodiments, cancers ofB-cell origin include, without limitation, B-lineage acute lymphoblasticleukemia, B-cell chronic lymphocytic leukemia, and B-cell non-Hodgkin'slymphoma.

2.5 Methods for Producing Recombinant AAV Vectors

In some embodiments, the invention provides recombinant AAV vectors foruse in the methods of the invention. Recombinant AAV vectors aretypically produced in mammalian cell lines such as HEK-293. Because theviral cap and rep genes are removed from the vector to prevent itsself-replication to make room for the therapeutic gene(s) to bedelivered (e.g. the endonuclease gene), it is necessary to provide thesein trans in the packaging cell line. In addition, it is necessary toprovide the “helper” (e.g. adenoviral) components necessary to supportreplication (Cots D, Bosch A, Chillon M (2013) Curr. Gene Ther. 13(5):370-81). Frequently, recombinant AAV vectors are produced using atriple-transfection in which a cell line is transfected with a firstplasmid encoding the “helper” components, a second plasmid comprisingthe cap and rep genes, and a third plasmid comprising the viral ITRscontaining the intervening DNA sequence to be packaged into the virus.Viral particles comprising a genome (ITRs and intervening gene(s) ofinterest) encased in a capsid are then isolated from cells byfreeze-thaw cycles, sonication, detergent, or other means known in theart. Particles are then purified using cesium-chloride density gradientcentrifugation or affinity chromatography and subsequently delivered tothe gene(s) of interest to cells, tissues, or an organism such as ahuman patient.

Because recombinant AAV particles are typically produced (manufactured)in cells, precautions must be taken in practicing the current inventionto ensure that the site-specific endonuclease is NOT expressed in thepackaging cells. Because the viral genomes of the invention comprise arecognition sequence for the endonuclease, any endonuclease expressed inthe packaging cell line will be capable of cleaving the viral genomebefore it can be packaged into viral particles. This will result inreduced packaging efficiency and/or the packaging of fragmented genomes.Several approaches can be used to prevent endonuclease expression in thepackaging cells, including:

-   -   1. The endonuclease can be placed under the control of a        tissue-specific promoter that is not active in the packaging        cells. For example, if a viral vector is developed for delivery        of (an) endonuclease gene(s) to muscle tissue, a muscle-specific        promoter can be used. Examples of muscle-specific promoters        include C5-12 (Liu, et al. (2004) Hum Gene Ther. 15:783-92), the        muscle-specific creatine kinase (MCK) promoter (Yuasa, et        al. (2002) Gene Ther. 9:1576-88), or the smooth muscle 22 (SM22)        promoter (Haase, et al. (2013) BMC Biotechnol. 13:49-54).        Examples of CNS (neuron)-specific promoters include the NSE,        Synapsin, and MeCP2 promoters (Lentz, et al. (2012) Neurobiol        Dis. 48:179-88). Examples of liver-specific promoters include        albumin promoters (such as Palb), human α1-antitrypsin (such as        PalAT), and hemopexin (such as Phpx) (Kramer, M G et al., (2003)        Mol. Therapy 7:375-85). Examples of eye-specific promoters        include opsin, and corneal epithelium-specific K12 promoters        (Martin K R G, Klein R L, and Quigley H A (2002) Methods (28):        267-75) (Tong Y, et al., (2007) J Gene Med, 9:956-66). These        promoters, or other tissue-specific promoters known in the art,        are not highly-active in HEK-293 cells and, thus, will not        expected to yield significant levels of endonuclease gene        expression in packaging cells when incorporated into viral        vectors of the present invention. Similarly, the viral vectors        of the present invention contemplate the use of other cell lines        with the use of incompatible tissue specific promoters (i.e.,        the well-known HeLa cell line (human epithelial cell) and using        the liver-specific hemopexin promoter). Other examples of tissue        specific promoters include: synovial sarcomas PDZD4        (cerebellum), C6 (liver), ASBS (muscle), PPP1R12B (heart),        SLC5A12 (kidney), cholesterol regulation APOM (liver), ADPRHL1        (heart), and monogenic malformation syndromes TP73L (muscle).        (Jacox E, et al., (2010) PLoS One v.5(8):e12274).    -   2. Alternatively, the vector can be packaged in cells from a        different species in which the endonuclease is not likely to be        expressed. For example, viral particles can be produced in        microbial, insect, or plant cells using mammalian promoters,        such as the well-known cytomegalovirus- or SV40 virus-early        promoters, which are not active in the non-mammalian packaging        cells. In a preferred embodiment, viral particles are produced        in insect cells using the baculovirus system as described by        Gao, et al. (Gao, H., et al. (2007) J. Biotechnol.        131(2):138-43). An endonuclease under the control of a mammalian        promoter is unlikely to be expressed in these cells (Airenne, K        J, et al. (2013) Mol. Ther. 21(4):739-49). Moreover, insect        cells utilize different mRNA splicing motifs than mammalian        cells. Thus, it is possible to incorporate a mammalian intron,        such as the human growth hormone (HGH) intron or the SV40 large        T antigen intron, into the coding sequence of an endonuclease.        Because these introns are not spliced efficiently from pre-mRNA        transcripts in insect cells, insect cells will not express a        functional endonuclease and will package the full-length genome.        In contrast, mammalian cells to which the resulting recombinant        AAV particles are delivered will properly splice the pre-mRNA        and will express functional endonuclease protein. Haifeng Chen        has reported the use of the HGH and SV40 large T antigen introns        to attenuate expression of the toxic proteins barnase and        diphtheria toxin fragment A in insect packaging cells, enabling        the production of recombinant AAV vectors carrying these toxin        genes (Chen, H (2012) Mol Ther Nucleic Acids. 1(11): e57).    -   3. The endonuclease gene can be operably linked to an inducible        promoter such that a small-molecule inducer is required for        endonuclease expression. Examples of inducible promoters include        the Tet-On system (Clontech; Chen H., et al., (2015) BMC        Biotechnol. 15(1):4)) and the RheoSwitch system (Intrexon; Sowa        G., et al., (2011) Spine, 36(10): E623-8). Both systems, as well        as similar systems known in the art, rely on ligand-inducible        transcription factors (variants of the Tet Repressor and        Ecdysone receptor, respectively) that activate transcription in        response to a small-molecule activator (Doxycycline or Ecdysone,        respectively). Practicing the current invention using such        ligand-inducible transcription activators includes: 1) placing        the endonuclease gene under the control of a promoter that        responds to the corresponding transcription factor, the        endonuclease gene having (a) binding site(s) for the        transcription factor; and 2) including the gene encoding the        transcription factor in the packaged viral genome The latter        step is necessary because the endonuclease will not be expressed        in the target cells or tissues following recombinant AAV        delivery if the transcription activator is not also provided to        the same cells. The transcription activator then induces        endonuclease gene expression only in cells or tissues that are        treated with the cognate small-molecule activator. This approach        is advantageous because it enables endonuclease gene expression        to be regulated in a spatio-temporal manner by selecting when        and to which tissues the small-molecule inducer is delivered.        However, the requirement to include the inducer in the viral        genome, which has significantly limited carrying capacity,        creates a drawback to this approach.    -   4. In another preferred embodiment, recombinant AAV particles        are produced in a mammalian cell line that expresses a        transcription repressor that prevents expression of the        endonuclease. Transcription repressors are known in the art and        include the Tet-Repressor, the Lac-Repressor, the Cro repressor,        and the Lambda-repressor. Many nuclear hormone receptors such as        the ecdysone receptor also act as transcription repressors in        the absence of their cognate hormone ligand. To practice the        current invention, packaging cells are transfected/transduced        with a vector encoding a transcription repressor and the        endonuclease gene in the viral genome (packaging vector) is        operably linked to a promoter that is modified to comprise        binding sites for the repressor such that the repressor silences        the promoter. The gene encoding the transcription repressor can        be placed in a variety of positions. It can be encoded on a        separate vector; it can be incorporated into the packaging        vector outside of the ITR sequences; it can be incorporated into        the cap/rep vector or the adenoviral helper vector; or, most        preferably, it can be stably integrated into the genome of the        packaging cell such that it is expressed constitutively. Methods        to modify common mammalian promoters to incorporate        transcription repressor sites are known in the art. For example,        Chang and Roninson modified the strong, constitutive CMV and RSV        promoters to comprise operators for the Lac repressor and showed        that gene expression from the modified promoters was greatly        attenuated in cells expressing the repressor (Chang B D, and        Roninson I B (1996) Gene 183:137-42). The use of a non-human        transcription repressor ensures that transcription of the        endonuclease gene will be repressed only in the packaging cells        expressing the repressor and not in target cells or tissues        transduced with the resulting recombinant AAV vector.

2.6 Recombinant Meganuclease Variants

Embodiments of the invention encompass the recombinant meganucleasesdescribed herein, and variants thereof. Further embodiments of theinvention encompass isolated polynucleotides comprising a nucleic acidsequence encoding the recombinant meganucleases described herein, andvariants of such polynucleotides.

As used herein, “variants” is intended to mean substantially similarsequences. A “variant” polypeptide is intended to mean a polypeptidederived from the “native” polypeptide by deletion or addition of one ormore amino acids at one or more internal sites in the native proteinand/or substitution of one or more amino acids at one or more sites inthe native polypeptide. As used herein, a “native” polynucleotide orpolypeptide comprises a parental sequence from which variants arederived. Variant polypeptides encompassed by the embodiments arebiologically active. That is, they continue to possess the desiredbiological activity of the native protein; i.e., the ability torecognize and cleave recognition sequences found in the human T cellreceptor alpha constant region (SEQ ID NO:1), including the TRC 1-2recognition sequence (SEQ ID NO:3), the TRC 3-4 recognition sequence(SEQ ID NO:4), and the TRC 7-8 recognition sequence (SEQ ID NO:5). Suchvariants may result, for example, from human manipulation. Biologicallyactive variants of a native polypeptide of the embodiments (e.g., SEQ IDNOs:8-32), or biologically active variants of the recognition half-sitebinding subunits described herein (e.g., SEQ ID NOs:33-82), will have atleast about 40%, about 45%, about 50%, about 55%, about 60%, about 65%,about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%,or about 99%, sequence identity to the amino acid sequence of the nativepolypeptide or native subunit, as determined by sequence alignmentprograms and parameters described elsewhere herein. A biologicallyactive variant of a polypeptide or subunit of the embodiments may differfrom that polypeptide or subunit by as few as about 1-40 amino acidresidues, as few as about 1-20, as few as about 1-10, as few as about 5,as few as 4, 3, 2, or even 1 amino acid residue.

The polypeptides of the embodiments may be altered in various waysincluding amino acid substitutions, deletions, truncations, andinsertions. Methods for such manipulations are generally known in theart. For example, amino acid sequence variants can be prepared bymutations in the DNA. Methods for mutagenesis and polynucleotidealterations are well known in the art. See, for example, Kunkel (1985)Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods inEnzymol. 154:367-382; U.S. Pat. No. 4,873,192; Walker and Gaastra, eds.(1983) Techniques in Molecular Biology (MacMillan Publishing Company,New York) and the references cited therein. Guidance as to appropriateamino acid substitutions that do not affect biological activity of theprotein of interest may be found in the model of Dayhoff et al. (1978)Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found.,Washington, D.C.), herein incorporated by reference. Conservativesubstitutions, such as exchanging one amino acid with another havingsimilar properties, may be optimal.

A substantial number of amino acid modifications to the DNA recognitiondomain of the wild-type I-CreI meganuclease have previously beenidentified (e.g., U.S. Pat. No. 8,021,867) which, singly or incombination, result in recombinant meganucleases with specificitiesaltered at individual bases within the DNA recognition sequencehalf-site, such that the resulting rationally-designed meganucleaseshave half-site specificities different from the wild-type enzyme. Table4 provides potential substitutions that can be made in a recombinantmeganuclease monomer or subunit to enhance specificity based on the basepresent at each half-site position (−1 through −9) of a recognitionhalf-site.

TABLE 4 Favored Sense-Strand Base Posn. A C G T NT A/C A/G C/T G/T A/G/TA/C/G/T −1 Y75 R70* K70 Q70* T46* G70 L75* H75* E70* C70 A70 C75* R75*E75* L70 S70 Y139* H46* E46* Y75* G46* C46* K46* D46* Q75* A46* R46*H75* H139 Q46* H46* −2 Q70 E70 H70 Q44* C44* T44* D70 D44* A44* K44*E44* V44* R44* I44* L44* N44* −3 Q68 E68 R68 M68 H68 Y68 K68 C24* F68C68 I24* K24* L68 R24* F68 −4 A26* E77 R77 S77 S26* Q77 K26* E26* Q26*−5 E42 R42 K28* C28* M66 Q42 K66 −6 Q40 E40 R40 C40 A40 S40 C28* R28*I40 A79 S28* V40 A28* C79 H28* I79 V79 Q28* −7 N30* E38 K38 I38 C38 H38Q38 K30* R38 L38 N38 R30* E30* Q30* −8 F33 E33 F33 L33 R32* R33 Y33 D33H33 V33 I33 F33 C33 −9 E32 R32 L32 D32 S32 K32 V32 I32 N32 A32 H32 C32Q32 T32

For polynucleotides, a “variant” comprises a deletion and/or addition ofone or more nucleotides at one or more sites within the nativepolynucleotide. One of skill in the art will recognize that variants ofthe nucleic acids of the embodiments will be constructed such that theopen reading frame is maintained. For polynucleotides, conservativevariants include those sequences that, because of the degeneracy of thegenetic code, encode the amino acid sequence of one of the polypeptidesof the embodiments. Variant polynucleotides include syntheticallyderived polynucleotides, such as those generated, for example, by usingsite-directed mutagenesis but that still encode a recombinantmeganuclease of the embodiments. Generally, variants of a particularpolynucleotide of the embodiments will have at least about 40%, about45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%,about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about94%, about 95%, about 96%, about 97%, about 98%, about 99% or moresequence identity to that particular polynucleotide as determined bysequence alignment programs and parameters described elsewhere herein.Variants of a particular polynucleotide of the embodiments (i.e., thereference polynucleotide) can also be evaluated by comparison of thepercent sequence identity between the polypeptide encoded by a variantpolynucleotide and the polypeptide encoded by the referencepolynucleotide.

The deletions, insertions, and substitutions of the protein sequencesencompassed herein are not expected to produce radical changes in thecharacteristics of the polypeptide. However, when it is difficult topredict the exact effect of the substitution, deletion, or insertion inadvance of doing so, one skilled in the art will appreciate that theeffect will be evaluated by screening the polypeptide for its ability topreferentially recognize and cleave recognition sequences found withinthe human T cell receptor alpha constant region gene (SEQ ID NO:1).

EXAMPLES

This invention is further illustrated by the following examples, whichshould not be construed as limiting. Those skilled in the art willrecognize, or be able to ascertain, using no more than routineexperimentation, numerous equivalents to the specific substances andprocedures described herein. Such equivalents are intended to beencompassed in the scope of the claims that follow the examples below.

Example 1 Characterization of Meganucleases that Recognize and CleaveTRC Recognition Sequences

1. Meganucleases that Recognize and Cleave the TRC 1-2 RecognitionSequence

Recombinant meganucleases (SEQ ID NOs:8-27), collectively referred toherein as “TRC 1-2 meganucleases,” were engineered to recognize andcleave the TRC 1-2 recognition sequence (SEQ ID NO:3), which is presentin the human T cell receptor alpha constant region. Each TRC 1-2recombinant meganuclease comprises an N-terminal nuclease-localizationsignal derived from SV40, a first meganuclease subunit, a linkersequence, and a second meganuclease subunit. A first subunit in each TRC1-2 meganuclease binds to the TRC1 recognition half-site of SEQ ID NO:3,while a second subunit binds to the TRC2 recognition half-site (see,FIG. 1A).

As illustrated in FIGS. 2 and 3, TRC1-binding subunits and TRC2-bindingsubunits each comprise a 56 base pair hypervariable region, referred toas HVR1 and HVR2, respectively. TRC1-binding subunits are identicaloutside of the HVR1 region except at position 80 or position 271(comprising a Q or E residue), and are highly conserved within the HVR1region. Similarly, TRC2-binding subunits are also identical outside ofthe HVR2 region except at position 80 or position 271 (comprising a Q orE residue), and at position 139 of meganucleases TRC 1-2x.87 EE, TRC1-2x.87 QE, TRC 1-2x.87 EQ, TRC 1-2x.87, and TRC 1-2x.163, whichcomprise an R residue (shaded grey and underlined). Like the HVR1region, the HVR2 region is also highly conserved.

The TRC1-binding regions of SEQ ID NOs:8-27 are illustrated in FIG. 2and are provided as SEQ ID NOs:33-52, respectively. Each of SEQ IDNOs:33-52 share at least 90% sequence identity to SEQ ID NO:33, which isthe TRC1-binding region of the meganuclease TRC 1-2x.87 EE (SEQ IDNO:8). TRC2-binding regions of SEQ ID NOs:8-27 are illustrated in FIG. 3and are provided as SEQ ID NOs:58-77, respectively. Each of SEQ IDNOs:58-77 share at least 90% sequence identity to SEQ ID NO:58, which isthe TRC2-binding region of the meganuclease TRC 1-2x.87 EE (SEQ IDNO:8).

2. Meganucleases that Recognize and Cleave the TRC 3-4 RecognitionSequence

Recombinant meganucleases (SEQ ID NOs:28 and 29), collectively referredto herein as “TRC 3-4 meganucleases,” were engineered to recognize andcleave the TRC 3-4 recognition sequence (SEQ ID NO:4), which is presentin the human T cell receptor alpha constant region. Each TRC 3-4recombinant meganuclease comprises an N-terminal nuclease-localizationsignal derived from SV40, a first meganuclease subunit, a linkersequence, and a second meganuclease subunit. A first subunit in each TRC3-4 meganuclease binds to the TRC3 recognition half-site of SEQ ID NO:4,while a second subunit binds to the TRC4 recognition half-site (see,FIG. 1A).

As illustrated in FIGS. 4 and 5, TRC3-binding subunits and TRC4-bindingsubunits each comprise a 56 base pair hypervariable region, referred toas HVR1 and HVR2, respectively. TRC3-binding subunits are identicaloutside of the HVR1 region except at position 80 or position 271(comprising a Q or E residue), and are highly conserved within the HVR1region. Similarly, TRC4-binding subunits are also identical outside ofthe HVR2 region except at position 80 or position 271 (comprising a Q orE residue), and are highly conserved within the HVR2 region.

The TRC3-binding regions of SEQ ID NOs:28 and 29 are illustrated in FIG.4 and are provided as SEQ ID NOs:53 and 54, respectively. SEQ ID NOs:53and 54 share 96.6% sequence identity. TRC4-binding regions of SEQ IDNOs:28 and 29 are illustrated in FIG. 5 and are provided as SEQ IDNOs:78 and 79, respectively. SEQ ID NOs:78 and 79 also share 96.6%sequence identity.

3. Meganucleases that Recognize and Cleave the TRC 7-8 RecognitionSequence

Recombinant meganucleases (SEQ ID NOs:30-32), collectively referred toherein as “TRC 7-8 meganucleases,” were engineered to recognize andcleave the TRC 7-8 recognition sequence (SEQ ID NO:5), which is presentin the human T cell receptor alpha constant region. Each TRC 7-8recombinant meganuclease comprises an N-terminal nuclease-localizationsignal derived from SV40, a first meganuclease subunit, a linkersequence, and a second meganuclease subunit. A first subunit in each TRC7-8 meganuclease binds to the TRC7 recognition half-site of SEQ ID NO:5,while a second subunit binds to the TRC8 recognition half-site (see,FIG. 1A).

As illustrated in FIGS. 6 and 7, TRC7-binding subunits and TRC8-bindingsubunits each comprise a 56 base pair hypervariable region, referred toas HVR1 and HVR2, respectively. TRC7-binding subunits are identicaloutside of the HVR1 region except at position 80 or position 271(comprising a Q or E residue), and are highly conserved within the HVR1region. Similarly, TRC8-binding subunits are also identical outside ofthe HVR2 region except at position 80 or position 271 (comprising a Q orE residue), and are highly conserved within the HVR2 region.

The TRC7-binding regions of SEQ ID NOs:30-32 are illustrated in FIG. 6and are provided as SEQ ID NOs:55-57, respectively. Each of SEQ IDNOs:55-57 share at least 90% sequence identity to SEQ ID NO:55, which isthe TRC7-binding region of the meganuclease TRC 7-8x.7 (SEQ ID NO:30).TRC8-binding regions of SEQ ID NOs:30-32 are illustrated in FIG. 7 andare provided as SEQ ID NOs:80-82, respectively. Each of SEQ ID NOs:80-82share at least 90% sequence identity to SEQ ID NO:80, which is theTRC8-binding region of the meganuclease TRC 7-8x.7 (SEQ ID NO:30).

4. Cleavage of Human T Cell Receptor Alpha Constant Region RecognitionSequences in a CHO Cell Reporter Assay

To determine whether TRC 1-2, TRC 3-4, and TRC 7-8 meganucleases couldrecognize and cleave their respective recognition sequences (SEQ IDNOs:3, 4, and 5, respectively), each recombinant meganuclease wasevaluated using the CHO cell reporter assay previously described (see,WO/2012/167192 and FIG. 8). To perform the assays, CHO cell reporterlines were produced which carried a non-functional Green FluorescentProtein (GFP) gene expression cassette integrated into the genome of thecells. The GFP gene in each cell line was interrupted by a pair ofrecognition sequences such that intracellular cleavage of eitherrecognition sequence by a meganuclease would stimulate a homologousrecombination event resulting in a functional GFP gene.

In CHO reporter cell lines developed for this study, one recognitionsequence inserted into the GFP gene was the TRC 1-2 recognition sequence(SEQ ID NO:3), the TRC 3-4 recognition sequence (SEQ ID NO:4), or theTRC 7-8 recognition sequence (SEQ ID NO:5). The second recognitionsequence inserted into the GFP gene was a CHO-23/24 recognitionsequence, which is recognized and cleaved by a control meganucleasecalled “CHO-23/24”. CHO reporter cells comprising the TRC 1-2recognition sequence and the CHO-23/24 recognition sequence are referredto herein as “TRC 1-2 cells.” CHO reporter cells comprising the TRC 3-4recognition sequence and the CHO-23/24 recognition sequence are referredto herein as “TRC 3-4 cells.” CHO reporter cells comprising the TRC 7-8recognition sequence and the CHO-23/24 recognition sequence are referredto herein as “TRC 7-8 cells.”

CHO reporter cells were transfected with plasmid DNA encoding theircorresponding recombinant meganucleases (e.g., TRC 1-2 cells weretransfected with plasmid DNA encoding TRC 1-2 meganucleases) or encodingthe CHO-23/34 meganuclease. In each assay, 4e⁵ CHO reporter cells weretransfected with 50 ng of plasmid DNA in a 96-well plate usingLipofectamine 2000 (ThermoFisher) according to the manufacturer'sinstructions. At 48 hours post-transfection, cells were evaluated byflow cytometry to determine the percentage of GFP-positive cellscompared to an untransfected negative control (TRC 1-2bs). As shown inFIG. 9, all TRC 1-2, TRC 3-4, and TRC 7-8 meganucleases were found toproduce GFP-positive cells in cell lines comprising their correspondingrecognition sequence at frequencies significantly exceeding the negativecontrol.

The efficacy of the TRC 1-2x.87 QE, TRC 1-2x.87 EQ, and TRC 1-2x.87 EEmeganucleases was also determined in a time-dependent manner. In thisstudy, TRC 1-2 cells (1e⁶) were electroporated with 1e⁶ copies ofmeganuclease mRNA per cell using a BioRad Gene Pulser Xcell according tothe manufacturer's instructions. At 1, 4, 6, 8, and 12 dayspost-transfection, cells were evaluated by flow cytometry to determinethe percentage of GFP-positive cells. As shown in FIG. 10, each TRC 1-2meganuclease exhibited high efficiency at 2 days post-transfection, withgreater than 50% GFP-positive cells observed. This effect persisted overthe 12 day period, with no evidence of cell toxicity observed.

5. Conclusions

These studies demonstrated that TRC 1-2 meganucleases, TRC 3-4meganucleases, and TRC 7-8 meganucleases encompassed by the inventioncan efficiently target and cleave their respective recognition sequencesin cells.

Example 2 Cleavage of TRC Recognition Sequences in T Cells andSuppression of Cell-Surface T Cell Receptor Expression 1. Cleavage ofthe TRC 1-2 Recognition Sequence in Jurkat Cells

This study demonstrated that TRC 1-2 meganucleases encompassed by theinvention could cleave the TRC 1-2 recognition sequence in Jurkat cells(an immortalized human T lymphocyte cell line). 1e⁶ Jurkat cells wereelectroporated with 8e⁶ copies of a given TRC 1-2 meganuclease mRNA percell using a BioRad Gene Pulser Xcell according to the manufacturer'sinstructions. At 72 hours post-transfection, genomic DNA (gDNA) washarvested from cells and a T7 endonuclease I (T7E) assay was performedto estimate genetic modification at the endogenous TRC 1-2 recognitionsequence (FIG. 11). In the T7E assay, the TRC 1-2 locus is amplified byPCR using primers that flank the TRC 1-2 recognition sequence. If thereare indels (random insertions or deletions) within the TRC 1-2 locus,the resulting PCR product will consist of a mix of wild-type alleles andmutant alleles. The PCR product is denatured and allowed to slowlyreanneal. Slow reannealing allows for the formation of heteroduplexesconsisting of wild-type and mutant alleles, resulting in mismatchedbases and/or bulges. The T7E1 enzyme cleaves at mismatch sites,resulting in cleavage products that can be visualized by gelelectrophoresis. FIG. 11 clearly demonstrates that thirteen differentversions of the TRC 1-2 meganucleases generated positive results in theT7E1 assay, indicating effective generation of indels at the endogenousTRC 1-2 recognition sequence.

To further examine the cleavage properties of TRC 1-2 meganucleases, adose-response experiment was performed in Jurkat cells. 1e⁶ Jurkat cellswere electroporated with either 3 μg or 1 μg of a given TRC 1-2meganuclease mRNA per cell using a BioRad Gene Pulser Xcell according tothe manufacturer's instructions. At 96-hours post-transfection, gDNA washarvested and the T7E1 assay was performed as described above. As seenin FIG. 12, fifteen different TRC 1-2 meganucleases showed cleavage atthe endogenous TRC 1-2 recognition site, including three differentversions of the TRC 1-2x.87 meganuclease. TRC 1-2x.87 EE workedespecially well, generating a strong signal in the T7E1 assay withlittle to no toxicity in Jurkat cells.

2. Cleavage of TRC 1-2 Recognition Sequence in Human T Cells

This study demonstrated that TRC 1-2 meganucleases encompassed by theinvention could cleave the TRC 1-2 recognition sequence in human T cellsobtained from a donor. CD3+ T cells were stimulated with anti-CD3 andanti-CD28 antibodies for 3 days, then electroporated with mRNA encodingthe TRC 1-2x.87 EE meganuclease using the Amaxa 4D-Nucleofector (Lonza)according to the manufacturer's instructions. At 3 days and 7 dayspost-transfection, gDNA was harvested and the T7E1 assay was performedas described above. FIG. 13A demonstrates that TRC 1-2x.87 EEeffectively introduced mutations in the endogenous TRC 1-2 recognitionsequence in human T cells, indicating that the meganuclease recognizedand cleaved the TRC 1-2 recognition sequence. The intensity of cleavageproducts does not appear to change between day 3 and day 7post-transfection, suggesting little or no toxicity due to the TRC1-2x.87 EE meganuclease. To determine whether the mutations at theendogenous TRC 1-2 recognition sequence were sufficient to eliminatesurface expression of the T cell receptor, cells were analyzed by flowcytometry using an anti-CD3 antibody. FIG. 13B shows that approximately50% of transfected T cells stained negative for CD3, indicating knockoutof the T cell receptor. The CD3 negative population did not changesignificantly between day 3 and day 7 post-transfection, furtherindicating little or no toxicity associated with the TRC 1-2x.87 EEmeganuclease, or the loss of T cell receptor expression.

To verify that loss of CD3 expression was due to mutations in the TRC1-2 recognition site, gDNA was harvested from transfected T cells andthe TRC 1-2 recognition site locus was amplified by PCR. PCR productswere cloned into the pCR-blunt vector using the Zero Blunt PCR cloningkit (Thermo Fisher) according to the manufacturer's instructions.Individual colonies were picked and mini-prepped plasmids weresequenced. FIG. 14 shows sequences of several representative deletionsthat were observed at the TRC 1-2 recognition sequence. The observedsequences are typical of deletions resulting from the non-homologous endjoining repair of DNA double-strand breaks generated by endonucleases.

In addition to TRC 1-2x.87 EE, other TRC 1-2 meganucleases were able toknockout the T cell receptor in human T cells, including TRC 1-2x.55,and TRC 1-2x.72, albeit to a lesser extent than knockout previouslyobserved for TRC 1-2x.87 EE (Tables 5 and 6). TRC 1-2x.72 Q47E carries amutation in the active site of the meganuclease (amino acid 47) andserves as a negative control.

TABLE 5 % CD3⁻ Cells Meganuclease Day 3 Day 6 TRC 1-2x.72 Q47E 0.38 1.1TRC 1-2x.55 3.11 10.84

TABLE 6 % CD3⁻ Cells Meganuclease Day 3 Day 5 TRC 1-2x.72 Q47E 0.29 0.4TRC 1-2x.72 2.09 4.19

3. Conclusions

These studies demonstrated that TRC 1-2 meganucleases encompassed by theinvention can recognize and cleave the TRC 1-2 recognition sequence inboth Jurkat cells (an immortalized T lymphocyte cell line) and in Tcells obtained from a human donor. Further, these studies demonstratedthat NHEJ occurs at the meganuclease cleavage site, as evidenced by theappearance of indels. Moreover, TRC 1-2 meganucleases were shown toreduce cell-surface expression of the T cell receptor on human T cellsobtained from a donor.

Example 3 Recombinant AAV Vectors For Introducing Exogenous NucleicAcids into Human T Cells 1. Recombinant AAV Vectors

In the present study, two recombinant AAV vectors (referred to as AAV405and AAV406) were designed to introduce an exogenous nucleic acidsequence, comprising an EagI restriction site, into the genome of humanT cells at the TRC 1-2 recognition sequence via homologousrecombination. Each recombinant AAV vector was prepared using atriple-transfection protocol, wherein a cell line is transfected with afirst plasmid encoding “helper” components (e.g., adenoviral) necessaryto support replication, a second plasmid comprising the cap and repgenes, and a third plasmid comprising the viral inverted terminalrepeats (ITRs) containing the intervening DNA sequence to be packagedinto the virus (e.g., the exogenous nucleic acid sequence) (see, Cots D,Bosch A, Chillon M (2013) Curr. Gene Ther. 13(5): 370-81). FIG. 15illustrates the general approach for using recombinant AAV vectors tointroduce an exogenous nucleic acid sequence into the cell genome at thenuclease cleavage site.

AAV405 was prepared using the plasmid illustrated in FIG. 16 (SEQ IDNO:107). As shown, the AAV405 plasmid generally comprises sequences fora 5′ ITR, a CMV enhancer and promoter sequence, a 5′ homology arm, anucleic acid sequence comprising the EagI restriction site, an SV40poly(A) signal sequence, a 3′ homology arm, and a 3′ ITR. AAV406 wasprepared using the plasmid illustrated in FIG. 17 (SEQ ID NO:108). Asshown, the AAV406 plasmid comprises similar sequences to those ofAAV405, but lacks the CMV enhancer and promoter sequences upstream ofthe 5′ homology arm. The present AAV studies further included the use ofan AAV vector encoding GFP (GFP-AAV), which was incorporated as apositive control for AAV transduction efficiency.

2. Introducing Exogenous Nucleic Acid Sequences into the TRC 1-2Recognition Sequence

To test whether AAV templates would be suitable for homology directedrepair (HDR) following generation of a double-strand break with TRC 1-2meganucleases, a series of experiments were performed using human Tcells. In the first experiment, the timing of electroporation with TRC1-2 RNA and transduction with recombinant AAV vectors was determined.Human CD3⁺ T cells were stimulated with anti-CD3 and anti-CD28antibodies for 3 days, then electroporated with mRNA encoding the TRC1-2x.87 EE meganuclease (1 μg) using the Amaxa 4D-Nucleofector (Lonza)according to the manufacturer's instructions. At either 2, 4, or 8 hourspost-transfection, cells were transduced with GFP-AAV (1e⁵ viral genomesper cell). Cells were analyzed by flow cytometry for GFP expression at72 hours post-transduction. As shown in FIG. 18, the highesttransduction efficiency was observed when cells were transduced at 2hours post-transfection (88% GFP-positive cells). Transductionefficiency decreased significantly as the time between transfection andtransduction increased, with 78% GFP-positive cells at 4 hours and 65%GFP-positive cells at 8 hours.

Having determined that efficient viral transduction occurred when cellswere transduced 2 hours post-transfection, the AAV405 and AAV406 vectorswere used as HDR templates in human T cells. CD3⁺ T cells werestimulated and transfected with 1 μg TRC 1-2x.87 EE mRNA as describedabove. At 2 hours post-transfection, cells were either transduced withAAV405 or AAV406 (1e⁵ viral genomes per cell). As transduction-onlycontrols, cells were mock transfected (with water) and transduced witheither AAV405 or AAV406 (1e⁵ viral genomes per cell). For ameganuclease-only control, cells were transfected with TRC 1-2x.87 EEand then mock transduced (with water) at 2 hours post-transfection.

To determine whether the AAV vectors served as HDR templates, gDNA washarvested from cells and the TRC 1-2 locus was amplified by PCR usingprimers that recognized sequences beyond the region of homology in theAAV vectors. PCR primers outside of the homology regions only allowedfor amplification of the T cell genome, not from the AAV vectors. PCRproducts were purified and digested with EagI. FIG. 19 shows cleavage ofthe PCR products amplified from cells that were transfected with TRC1-2x.87 EE and transduced with either AAV vector (see arrows),indicating insertion of the EagI site into the TRC 1-2 recognitionsequence. The PCR products from all of the control cell populations arenot cleaved by EagI, demonstrating that the insertion of the EagI siterequires creation of a DNA double-strand break by a TRC 1-2meganuclease.

To further define the insertion of the EagI site into human T cells,individual products from the bulk PCR product were examined. UndigestedPCR product generated from the above experiment was cloned into thepCR-blunt vector using the Zero Blunt PCR cloning kit (Thermo Fisher)according to the manufacturer's instructions. Colony PCR was performedusing M13 forward and reverse primers (pCR blunt contains M13 forwardand reverse priming sites flanking the insert) and a portion of PCRproducts from cells transfected with TRC 1-2x.87 EE and either AAV405 orAAV406 were analyzed by gel electrophoresis (FIGS. 20A and 21A,respectively). In both cases, there are a mix of full-length PCRproducts (approximately 1600 bp), smaller inserts, and some emptyplasmids (approximately 300 bp). In this assay, bands smaller thanfull-length but larger than empty plasmids are often times sequencescontaining large deletions within the TRC 1-2 recognition sequence. Inparallel, another portion of PCR products were digested with EagI todetermine the percent of clones that contain the EagI recognition siteinserted into the TRC 1-2 recognition sequence. FIGS. 20B and 21B showthat several PCR products were cleaved with EagI (e.g., FIG. 20B, secondrow, 6 lanes from the left), generating the expected fragments ofapproximately 700 and 800 bp. These gels allow for the estimation ofEagI insertion to be approximately 25% and 6% for AAV405 and AAV406,respectively (adjusted for empty vectors).

To confirm observations from gel electrophoresis of uncut PCR productsand digest with EagI, the remaining portion of each PCR product wassequenced. FIG. 22A shows sequences of several representative deletionsand insertions that were observed at the TRC 1-2 recognition sequence.These sequences are typical of sequences resulting from thenon-homologous end joining repair of DNA double-strand breaks generatedby endonucleases. All PCR products that were cleaved with EagI containedan EagI site inserted into the TRC 1-2 recognition sequence (FIG. 22B).

3. Enhanced AAV Transduction Efficiency

In light of the observation that AAV transduction was more efficientwhen it was carried out 2 hours post-transfection than when it wascarried out later, an experiment was performed to optimize the timing oftransfection and transduction. Human CD3⁺ T cells were stimulated withanti-CD3 and anti-CD28 antibodies for 3 days, then electroporated withthe TRC 1-2x.87 EE meganuclease (1 μg) using the Amaxa 4D-Nucleofector(Lonza) according to the manufacturer's instructions. Immediately aftertransfection or 2 hours post-transfection, cells were transduced withGFP-AAV (1e⁵ viral genomes per cell). Additionally, non-stimulated cellswere transduced with GFP-AAV (1e⁵ viral genomes per cell). At 72 hourspost-transduction, cells were analyzed by flow cytometry for GFPexpression. FIG. 23 shows that GFP-AAV transduction performed 2 hourspost-transfection resulted in 90% GFP-positive cells, but thattransduction immediately after transfection resulted in 98% GFP-positivecells. Resting T cells appeared refractive to AAV transduction, withapproximately 0% GFP-positive cells. Non-transduced cells also showedapproximately 0% GFP-positive cells.

4. Summary

These studies demonstrate that AAV vectors can be used in conjunctionwith recombinant meganucleases to incorporate an exogenous nucleic acidsequence into a cleavage site in the TCR alpha constant region viahomologous recombination.

Example 4 Recombinant AAV Vectors for Introducing Exogenous NucleicAcids Encoding a Chimeric Antigen Receptor in Human T Cells 1.Recombinant AAV Vectors

In the present study, two recombinant AAV vectors (referred to asAAV-CAR100 and AAV-CAR763) were designed to introduce an exogenousnucleic acid sequence, encoding a chimeric antigen receptor, into thegenome of human T cells at the TRC 1-2 recognition sequence viahomologous recombination. Each recombinant AAV vector was prepared usingthe triple-transfection protocol described previously.

AAV-CAR100 (also referred to herein as AAV408) was prepared using theplasmid illustrated in FIG. 24 (SEQ ID NO:109). As shown, the AAV-CAR100(AAV408) is designed for producing a self-complementary AAV vector, andgenerally comprises sequences for a 5′ ITR, a 5′ homology arm, a nucleicacid sequence encoding an anti-CD19 chimeric antigen receptor, an SV40poly(A) signal sequence, a 3′ homology arm, and a 3′ ITR. AAV-CAR763(also referred to herein as AAV412) was prepared using the plasmidillustrated in FIG. 25 (SEQ ID NO:110). As shown, the AAV-CAR763(AAV412) plasmid generally comprises the same sequences as AAV-CAR100(AAV408), but is designed for producing a single-stranded AAV vector.Because a single-stranded AAV vector can accommodate a larger payload,the 5′ homology arm and the 3′ homology arm are longer in AAV-CAR763(AAV412) than in AAV-CAR100 (AAV408). The present AAV studies willfurther include the use of an AAV vector encoding GFP (GFP-AAV), whichwill be incorporated as a positive control for AAV transductionefficiency.

2. Introducing a Chimeric Antigen Receptor Sequence into the TRC 1-2Recognition Sequence

Studies will be conducted to determine the efficiency of usingrecombinant AAV vectors to insert a chimeric antigen receptor sequenceinto the TCR alpha constant region gene while, simultaneously, knockingout cell-surface expression of the endogenous TCR receptor.

To confirm transduction efficiency, human CD3⁺ T cells will be obtainedand stimulated with anti-CD3 and anti-CD28 antibodies for 3 days, thenelectroporated with mRNA encoding the TRC 1-2x.87 EE meganuclease (1 μg)using the Amaxa 4D-Nucleofector (Lonza) according to the manufacturer'sinstructions. Cells will be transduced with GFP-AAV (1e⁵ viral genomesper cell) immediately after transfection as described above. Cells willbe analyzed by flow cytometry for GFP expression at 72 hourspost-transduction to determine transduction efficiency.

AAV-CAR100 (AAV408) and AAV-CAR763 (AAV412) vectors will then be used asHDR templates in human T cells for the insertion of the anti-CD19chimeric antigen receptor sequence. Human CD3⁺ T cells will bestimulated and transfected with 1 μg TRC 1-2x.87 EE mRNA as describedabove. Cells will then be transduced with AAV-CAR100 (AAV408) orAAV-CAR763 (AAV412) (1e⁵ viral genomes per cell) either immediatelyafter transfection or within 0-8 hours of transfection. Astransduction-only controls, cells will be mock transfected (with water)and transduced with either AAV-CAR100 (AAV408) or AAV-CAR763 (AAV412)(1e⁵ viral genomes per cell). For a meganuclease-only control, cellswill be transfected with mRNA encoding TRC 1-2x.87 EE and then mocktransduced (with water) immediately post-transfection.

Insertion of the chimeric antigen receptor sequence will be confirmed bysequencing of the cleavage site in the TCR alpha constant region gene.Cell-surface expression of the chimeric antigen receptor will beconfirmed by flow cytometry, using an anti-Fab or anti-CD19 antibody.Knockout of the endogenous T cell receptor at the cell surface will bedetermined by flow cytometry as previously described.

Example 5 Insertion and Expression of Chimeric Antigen Receptor

1. Insertion of Chimeric Antigen Receptor Sequence into the TRC 1-2Recognition Sequence

In the present study, we test whether AAV can provide HDR templates thatcan be used to insert a chimeric antigen receptor sequence into the TCRalpha constant region gene and, simultaneously, knock out cell-surfaceexpression of the endogenous TCR receptor. In the first experiment,human CD3+ T cells (1e⁶ cells) were stimulated and electroporated withmRNA encoding the TRC 1-2x.87 EE meganuclease (2 μg) as described above,then immediately transduced with AAV412 (1e⁵ viral genomes/cell). Ascontrols, cells were mock electroporated, then transduced with AAV412 orelectroporated with mRNA encoding TRC 1-2x.87EE, then mock transduced.An additional control of mock electroporated, mock transduced cells wasincluded.

A PCR-based assay was developed to determine whether the AAV HDRtemplate was utilized to repair double-strand breaks at the TRC 1-2recognition sequence. Three sets of primer pairs were used for PCRanalysis. The first set was designed to amplify a region with thehomology arms of AAV412. Since this first primer set (referred to as“Inside homolog arms/CAR region” in Table 7) lies within the homologyregion, it will either amplify the unmodified TRC 1-2 recognitionsequence locus of the genome (349 bp), the AAV412 vector input (2603bp), or the TRC 1-2 recognition sequence into which the CAR gene hasbeen inserted (2603 bp). The second primer set (referred to as “Outside5′ homology arm” in Table 7) includes one primer that anneals within theCAR region of the AAV412 HDR template, one primer that anneals in thehuman genome, outside of the 5′ homology arm of the AAV412 HDR templateand will amplify an 1872 bp fragment only if the CAR gene wassuccessfully inserted into the TRC 1-2 recognition sequence. The thirdprimer set (referred to as “Outside 3′ homology arm” in Table 7)includes one primer that anneals within the CAR region of the AAV412 HDRtemplate, and one primer that anneals in the human genome, outside ofthe 3′ homology arm of the AAV412 HDR template. Similarly to the secondprimer set, the third primer set will amplify an 1107 bp fragment onlyif the CAR gene was successfully inserted into the TRC 1-2 recognitionsequence. Taken together, PCR products from all three primer sets willindicate whether the CAR sequence is present in cells (primer set 1),and whether it has been inserted into the TRC 1-2 recognition sequence(primer sets 2 and 3).

On day 4 post-transduction cells were analyzed using the PCR primerpairs described above. Briefly, approximately 3,000 cells wereharvested, pelleted, and lysed and PCR was performed to determinewhether the CAR gene was inserted into the TRC 1-2 recognition sequence.PCR products were resolved on an agarose gel, shown in FIG. 26 (lanedescriptions can be found in Table 7). Lanes 1-3 are PCR products fromthe sample that was electroporated with mRNA encoding TRC 1-2x.87EE andmock transduced.

As expected, the first primer pair (“Inside homolog arms/CAR region”)amplified the unmodified TRC 1-2 recognition sequence locus, generatinga 349 bp band shown in lane 1. Lanes 2 and 3 correspond to primer pairsthat only generate a product if the CAR gene has been inserted into theTRC 1-2 recognition sequence, and do not show products. Lanes 7-9represent samples that were mock electroporated and mock transduced andshow the same bands as the TRC 1-2x.87EE mRNA only control describedabove. Lanes 4-6 show PCR products from the sample that waselectroporated with TRC 1-2x.87EE mRNA and transduced with AAV412. Lane4 shows two bands generated by the first primer pair (“Inside homologarms/CAR region”), indicating amplification of the unmodified TRC 1-2recognition sequence locus of the genome (349 bp) and the AAV412 vectorinput (2603 bp) or the TRC 1-2 recognition sequence into which the CARgene has been inserted (2603 bp). Lanes 5 and 6 show products generatedby the primer pairs that only amplify products if the CAR nucleic acidsequence has been inserted into the TRC 1-2x.87EE recognition site. Bothbands are the predicted size (1872 and 1107 bp, respectively). Lanes10-12 represent the sample that was mock electroporated and transducedwith AAV412. Lane 10 shows two bands generated by the first primer pair(“Inside homolog arms/CAR region”), indicating amplification of theunmodified TRC 1-2 recognition sequence locus of the genome (349 bp) andthe AAV412 vector input (2603 bp). Lanes 11 and 12 correspond to primerpairs that only generate a product if the CAR gene has been insertedinto the TRC 1-2 recognition sequence, and do not show products. Theabsence of bands in lanes 11 and 12 (which include primers outside ofthe homology arm) indicates that the 2603 bp band in lane 10 wasgenerated from amplification of the AAV412 input.

Taken together, the PCR analysis clearly demonstrates that CAR genes areintroduced into the TRC 1-2x.87EE recognition site when both TRC1-2x.87EE mRNA and AAV412 are present in cells. Thus, we conclude thatAAV412 serves to produce suitable HDR templates that can be used toinsert a CAR gene into the TRC 1-2x.87EE recognition sequence.

In a second experiment, human CD3⁺ T cells were stimulated andelectroporated with mRNA encoding the TRC 1-2x.87 EE meganuclease asdescribed above, then immediately transduced with increasing amounts ofAAV408 (0 μL, 3.125 μL, 6.25 μL, 12.5 μL, or 25 μL, which corresponds toapproximately 0, 3.125e³, 6.250e³, 1.25e⁴ and 2.5e⁴ viral genomes/cell).As controls, cells were mock electroporated, then transduced withincreasing amounts of AAV408. Additional controls included cells thatwere mock electroporated and mock transduced, as well as cells that wereelectroporated with TRC 1-2x.87EE mRNA then mock transduced. On day 4post-transduction, cells were harvested and analyzed as described above,but only using the primer pairs that amplified a product only if the CARgene has been inserted into the TRC 1-2 recognition sequence. PCRproducts were resolved on agarose gels, shown in FIG. 27. FIG. 27A showsthe PCR products generated using the primer pair described above(“Outside 5′ homology arm”) which only amplifies a product on the 5′ endof the TRC 1-2 recognition sequence locus if the CAR gene has beeninserted into that locus. FIG. 27B shows the PCR products generatedusing the primer pair described above (“Outside 3′ homology arm”) whichonly amplifies a product on the 3′ end of the TRC 1-2 recognitionsequence locus if the CAR gene has been inserted into that locus. Lanedescriptions can be found in Table 8. Lanes 1-5 in both FIGS. 27A and27B represent the samples that were either mock electroporated or mockelectroporated then mock transduced. No PCR products are visible in mockelectroporated cells, indicating the HDR templates produced by AAV408are unable to insert the CAR gene into the TRC 1-2 recognition sequencein the absence of TRC 1-2x.87EE mRNA. Lane 6 represents the sample thatwas electroporated with TRC 1-2x.87EE mRNA and mock transduced. No PCRproducts are visible, indicating that the CAR gene had not been insertedinto the TRC 1-2 recognition sequence. Lanes 7-10 represent samples thatwere electroporated with TRC 1-2x.87EE mRNA and transduced withincreasing amounts of AAV408. The appropriately sized bands for each PCRare evident, indicating that AAV408 can produce HDR donors for repair ofthe TRC 1-2 recognition sequence, resulting in insertion of the CARgene.

TABLE 7 Virus (100 k Sample Nucleofection MOI) PCR Product Size  1TRC1-2x87EE — Inside homolog arms/ Genomic = 349 bp CAR region +CD19 =2603 bp  2 TRC1-2x87EE — Outside 5′ homology arm 1872 bp  3 TRC1-2x87EE— Outside 3′ homology arm 1107 bp  4 TRC1-2x87EE AAV412 Inside homologarms/ Genomic = 349 bp CAR region +CD19 = 2603 bp  5 TRC1-2x87EE AAV412Outside 5′ homology arm 1872 bp  6 TRC1-2x87EE AAV412 Outside 3′homology arm 1107 bp  7 Mock (Water) Inside homolog arms/ Genomic = 349bp CAR region +CD19 = 2603 bp  8 Mock (Water) Outside 5′ homology arm1872 bp  9 Mock (Water) Outside 3′ homology arm 1107 bp 10 Mock (Water)AAV412 Inside homolog arms/ Genomic = 349 bp CAR region +CD19 = 2603 bp11 Mock (Water) AAV412 Outside 5' homology arm 1872 bp 12 Mock (Water)AAV412 Outside 3' homology arm 1107 bp

TABLE 8 Virus (AAV408) Sample Nucleofection μL  1 Mock (Water) 0  2 Mock(Water) 3.125  3 Mock (Water) 6.25  4 Mock (Water) 12.5  5 Mock (Water)25  6 TRC1-2x87EE 0  7 TRC1-2x87EE 3.125  8 TRC1-2x87EE 6.25  9TRC1-2x87EE 12.5 10 TRC1-2x87EE 25

The PCR-based assays described above are useful in determining whetherthe CAR gene had been inserted into the TRC 1-2 recognition sequence,but do not give information on efficiency. To determine the efficiencyof CAR insertion, we developed a digital PCR-based assay (schematicshown in FIG. 28A). In this assay, two primer sets are used. The firstset amplifies an irrelevant gene sequence and serves a referencesequence to control for template number. The second set consists of oneprimer that anneals within the CAR gene and one primer that annealsoutside of the 3′ homology arm, such that a product is only amplified ifthe CAR gene has been inserted into the TRC 1-2 recognition sequence. AVIC-labeled probe anneals within the amplicon generated from the firstprimer set and FAM-labeled probe anneals within the amplicon generatedby the second set of primers. By dividing the number of ampliconsdetected by the FAM-labeled probe to the number of reference sequenceamplicons detected by the VIC-labeled probe, it is possible toaccurately quantitate the percent of TRC 1-2 recognition sequence locithat were modified by insertion of the CAR gene.

FIG. 28B shows the results of the digital PCR assay for samples thatwere either mock electroporated then transduced, electroporated with TRC1-2x.87EE mRNA then mock transduced, or electroporated with TRC1-2x.87EE mRNA then transduced with increasing amounts of AAV408.Digital PCR was performed using genomic DNA isolated from cellsapproximately 1 week post-transduction. Consistent with the observationsfrom the PCR described in FIG. 27, both control samples (transductiononly or electroporation only) were found to have 0% CAR gene insertedinto the TRC 1-2x.87EE recognition sequence. Samples that wereelectroporated with mRNA encoding TRC 1-2x.87EE then transduced withincreasing amounts of AAV408 were found to have between approximately1.5% and 7%. The assay was performed on two different instruments(labeled QX200 and QS3D) and showed remarkable agreement, demonstratingthe sensitivity and precision of this digital PCR-based assay.

2. Expression of Anti-CD19 Chimeric Antigen Receptor on T Cells

In addition to determining whether CAR insertion occurred at themolecular level, we sought to determine the expression level of theanti-CD19 chimeric antigen receptor in cells that had the CAR geneinserted into the TRC 1-2 recognition sequence using AAV408 as the HDRtemplate. Additionally, we examined the efficiency in which insertion ofthe CAR into the TRC 1-2x.87EE recognition sequence resulted in knockoutof the T cell receptor. Samples described above and analyzed in FIGS. 27and 28 were also analyzed for CAR and CD3 expression by flow cytometry.Approximately 4 days post-transduction, cells were labeled withantibodies that recognize the anti-CD19 CAR (anti-Fab-Alexa647) or CD3(CD3⁻BB515) and analyzed by flow cytometry. FIG. 29A shows flowcytometry plots, with anti-CAR labeling shown on the Y axis and anti-CD3labeling shown on the X axis. Cells that were mock electroporated andmock transduced (MOI-0) were overwhelmingly CD3⁺/CAR⁻ (the lower rightquadrant, 98.7%). Cells that were mock electroporated then transducedwith increasing amounts of AAV408 looked essentially identical to thecontrol cells, with the CD3⁺/CAR⁻ populations at 98.8%, 99, 99%, and99.1%. Thus we conclude that the AAV408 virus alone is not drivingdetectable levels of CAR expression, nor is it capable of disruptingexpression of the T cell receptor.

FIG. 29B shows flow cytometry plots for samples that were eitherelectroporated with mRNA encoding TRC 1-2x.87EE then mock transduced orcells that were electroporated with TRC 1-2x.87EE then transduced withincreasing amounts of AAV408. Cells that were electroporated then mocktransduced show 47.1% CD3⁻ cells, indicating efficient knockout of the Tcell receptor complex. Background labeling with the anti-CD19 CAR wasvery low, with 0.6% in the CD3⁻ population and 0.78% in the CD3⁺population. Samples that were electroporated with mRNA encoding TRC1-2x.87EE then transduced with increasing amounts of AAV408 showed CARlabeling in the CD3⁻ population, ranging from 2.09% to 5.9%. There wasalso a slight increase in CAR labeling in the CD3⁺ population, rangingfrom 1.08% to 1.91%. We did not determine the cause of the increase inCARP cells in the CD3⁺ population, although it is possible that the CARwas inserted into the non-expressed T cell receptor allele (only oneallele of the T cell receptor alpha chain is expressed and incorporatedinto the T cell receptor complex).

These data correlated well with the quantitative digital PCR-based assaydescribed above. For example, at the highest MOI of AAV408 (2.5e⁴ viralgenomes/cell), the digital PCR assay showed approximately 6% CARinsertion, and the flow cytometry assay showed 5.9% CAR⁺/CD3⁻ cells. Ifone takes into account the CAR⁺/CD3⁺ population, the data are stillquite comparable, with the flow cytometry assay showing approximately7.8% CARP compared to 6% by digital PCR.

Example 6 Characterization of Additional AAV Vectors

1. Insertion of a Chimeric Antigen Receptor Sequence into the TRC 1-2Recognition Sequence

Having shown that AAV vectors could provide suitable HDR templates toinsert CAR genes into the TRC 1-2x.87EE recognition sequence, we soughtto optimize the configuration of the AAV vector. We generated a vectorthat could be used to produce self-complementary AAV genomes thatincluded the CAR gene expression cassette driven by a JeT promoter,flanked by short regions of homology to the TRC 1-2 recognition sequencelocus and AAV ITRs. This vector is referred to as AAV421 (FIG. 30; SEQID NO:123). Short homology arms were necessary due to limited packagingcapacity of self-complementary AAV. Additionally, we generated a vectorthat could be used to produce single-strand AAV genomes that includesthe CAR gene expression cassette driven by a CMV promoter, flanked bylong homology arms and AAV ITRs. This vector is referred to as AAV422(FIG. 31; SEQ ID NO:124). Since single-strand AAV genomes have a largercargo capacity, we were able to utilize longer homology arms than in theself-complementary vector.

To test whether AAV421 and AAV422 were useful to target insertion of theCAR gene into the TRC 1-2 recognition sequence, several experimentssimilar to those described above were carried out in human CD3⁺ T cells.In a first experiment, human CD3⁺ T cells (1e⁶ cells) were either mockelectroporated then transduced with increasing amounts of AAV421 or 422,or electroporated with TRC 1-2x.87EE mRNA (2 μg) then transduced withincreasing amounts of AAV421 or AAV422. AAV422 MOIs were significantlyhigher than AAV421 in this experiment than in the experiments describedabove (approximate MOIs were 1.25e⁴, 2.5e⁴, 5e⁴ and 1e⁵ viralgenomes/cell) because earlier experiments with AAV408 suggested thathigher MOIs would result in more efficient CAR insertion. The AAV421virus stock was not concentrated enough to allow for titerssignificantly higher than in the experiments described earlier. Ascontrols, cells were electroporated (mock or with TRC 1-2x.87EE mRNA)then mock transduced. As an additional component to this experiment, a“large scale” condition was performed, in which 10e⁶ cells (10 timesmore than a typical experiment) were electroporated with TRC 1-2x.87EEmRNA then transduced with AAV422 (2.5e⁴ viral genomes/cell). Lastly, wealso tested a second virus stock of AAV421 to compare to the primaryvirus stock.

Insertion of the CAR was determined by PCR as described above, usingprimer pairs that only amplify products if the CAR gene has beeninserted into the TRC 1-2x.87EE recognition sequence. PCR was resolvedby agarose gel, shown in FIGS. 32A and 32B (lane descriptions can befound in Tables 9 and 10). Sample 1 in FIG. 32A was mock electroporatedthen mock transduced, and samples 2-5 were mock electroporated thentransduced with AAV421. The gel shows that none of these samplesgenerated PCR products, indicating that AAV421, in the absence of TRC1-2x.87EE mRNA, is unable to drive insertion of the CAR gene into theTRC 1-2 recognition sequence. Additionally, the control sample that waselectroporated with TRC 1-2x.87EE mRNA then mock transduced (sample 6),did not show any PCR products. Samples 7-10 in FIG. 32A wereelectroporated with TRC 1-2x.87EE mRNA, then transduced with increasingamounts of AAV421. The gel shows PCR bands for products extending beyondboth the 5′ and 3′ homology arm (the two bands under each samplenumber), demonstrating integration of the CAR gene into the TRC 1-2recognition sequence. Lastly in FIG. 32A, lanes 11 and 12 representsamples that were electroporated with TRC 1-2x.87EE mRNA then transducedwith AAV422, either starting with 1e⁶ or 10e⁶ cells/sample,respectively. The presence of both PCR bands (larger in the first set,because different primer was used to account for a longer homology arm)indicate successful insertion of the CAR gene into the TRC 1-2recognition sequence.

Sample 1 in FIG. 32B was mock electroporated then mock transduced, andsamples 2-5 were mock electroporated then transduced with increasingamounts of AAV422 (Table 10). The gel shows that none of these samplesgenerated PCR products, indicating that AAV422, in the absence of TRC1-2x.87EE mRNA, is unable to drive insertion of the CAR gene into theTRC 1-2 recognition sequence. Samples 7-10 in FIG. 32B wereelectroporated with TRC 1-2x.87EE mRNA, then transduced with increasingamounts of AAV422. The gel shows PCR bands for products extending beyondboth the 5′ and 3′ homology arm, demonstrating integration of the CARgene into the TRC 1-2 recognition sequence. Lastly, sample 11 representsthe sample that was electroporated with TRC 1-2x.87EE mRNA thentransduced with a AAV421 from a different virus stock than samples shownin FIG. 32A. The presence of bands indicate insertion of the CAR geneinto the TRC 1-2 recognition sequence and confirms reproducibilitybetween different virus stocks. Taken together, FIG. 32 clearlydemonstrates that both AAV421 and AAV422 are capable of generating HDRtemplates suitable for inserting the CAR gene into the TRC 1-2recognition sequence.

TABLE 9 AAV μl MOI Sample Nucleofection Virus AAV (approximate)  1 Mock(Water) 421 0 0  2 Mock (Water) 421 3.125 3906  3 Mock (Water) 421 6.257813  4 Mock (Water) 421 12.5 15625  5 Mock (Water) 421 25 31250  6TRC1-2x87EE 421 0 0  7 TRC1-2x87EE 421 3.125 3906  8 TRC1-2x87EE 4216.25 7813  9 TRC1-2x87EE 421 12.5 15625 10 TRC1-2x87EE 421 25 31250 11TRC1-2x87EE 422 6.25 25000 12 TRC1-2x87EE 422 62.5 25000 Large Scale

TABLE 10 AAV μl MOI Sample Nucleofection Virus AAV (approximate)  1 Mock(Water) 422 0 0  2 Mock (Water) 422 3.125 12500  3 Mock (Water) 422 6.2525000  4 Mock (Water) 422 12.5 50000  5 Mock (Water) 422 25 100000  6TRC1-2x87EE 422 0 0  7 TRC1-2x87EE 422 3.125 12500  8 TRC1-2x87EE 4226.25 25000  9 TRC1-2x87EE 422 12.5 50000 10 TRC1-2x87EE 422 25 100000 11TRC1-2x87EE  421B 25 10000

2. Expression of Anti-CD19 Chimeric Antigen Receptor on T Cells UsingAAV421

Here, we sought to determine the expression level of the anti-CD19chimeric antigen receptor in cells that had the CAR gene inserted intothe TRC 1-2 recognition sequence using AAV421. Samples described aboveand analyzed in FIG. 32A were also analyzed for CAR and CD3 expressionby flow cytometry. Approximately 4 days post-transduction, cells werelabeled with antibodies that recognize the anti-CD19 CAR or CD3 andanalyzed by flow cytometry. FIG. 33A shows flow cytometry plots forcells that were mock electroporated and transduced with AAV421, alongwith control cells that were mock electroporated and mock transduced.Cells that were mock electroporated and mock transduced (MOI-0) wereoverwhelmingly CD3⁺/CAR⁻ (the lower right quadrant, 98.8%). Cells thatwere mock electroporated then transduced with increasing amounts ofAAV421 looked essentially identical to the control cells, with theCD3⁺/CAR⁻ populations at 98.8%, 98.6%, 98.8% and 97.9%. Thus, weconclude that the AAV421 virus alone is not driving detectable levels ofCAR expression, nor is it capable of disrupting expression of the T cellreceptor.

FIG. 33B shows flow cytometry plots for samples that were eitherelectroporated with TRC 1-2x.87EE mRNA then mock transduced or cellsthat were electroporated with TRC 1-2x.87EE then transduced withincreasing amounts of AAV421. Cells that were electroporated then mocktransduced show 56.7% CD3⁻ cells, indicating efficient knockout of the Tcell receptor complex. Background labeling with the anti-CD19 CAR wasvery low, with 0.48% in the CD3⁻ population and 0.36% in the CD3⁺population. Samples that were electroporated with mRNA encoding TRC1-2x.87EE then transduced with increasing amounts of AAV412 showedsignificant amounts of CAR labeling in the CD3⁻ population, ranging from4.99% to 13.4%. There was also a slight increase in CAR labeling in theCD3⁺ population, ranging from 1.27% to 3.95%. As mentioned above, it ispossible that the CAR gene was inserted into the non-expressed T cellreceptor allele. Also in contrast to experiments with AAV408, the CARPpopulation was much better defined, with a higher mean fluorescenceintensity, suggesting that the JeT promoter drives higher expressionthan the eF1α core promoter.

While evaluating insertion of the CAR gene using AAV421 in conjunctionwith TRC 1-x.87EE, we sought to determine a method that would allow usto preferentially expand and enrich the CD3⁻/CAR⁺ population. From theexperiment described above and shown in FIG. 33, we used cells that wereelectroporated with TRC 1-2x.87EE mRNA (2 μg) then transduced withAAV421 (3.13e⁴ viral genomes/cell). Control samples were mockelectroporated and mock transduced, mock electroporated and transducedwith AAV421, or electroporated with TRC 1-2x.87EE and mock transducedtaken from the experiment described above and shown in FIG. 33. As acontrol enrichment and expansion process, these cells were incubated for6 days in complete growth medium supplemented with IL-7 and IL-15 (bothat 10 ng/mL). Cells were then labeled with antibodies against theanti-CD19 CAR and CD3 and analyzed by flow cytometry (FIG. 34A). Cellsthat were mock electroporated and mock transduced showed low levels ofbackground staining in the CD3⁻/CAR⁺ quadrant (0.13%). The CD3⁻/CAR⁺population was essentially the same in samples that were either mockelectroporated then transduced with AAV or electroporated with TRC1-2x.87EE mRNA then mock transduced (0.16% and 0.55%, respectively).Cells that were electroporated with TRC 1-2x.87EE mRNA and mocktransduced had a CD3⁻/CAR⁻ population of 53.2%, very close to the amountstained in the first part of this experiment shown in FIG. 33B (56.7%).Cells that were electroporated with TRC 1-2x.87EE and transduced withAAV showed 12.6% CD3⁻/CAR⁺ cells, almost identical to the originallabeling of these cells shown in FIG. 33 (13.4%), demonstrating thatmixture of IL-7 and IL-15 is insufficient to enrich or expand thespecific CD3⁻/CAR⁺ cell population.

We next sought to enrich for the CD3⁻/CAR⁺ population in anantigen-specific manner by incubating the 4 samples described above withIM-9 cells, which present CD19 on the cell surface. IM-9 cells wereinactivated by pre-treatment with mitomycin C and incubated with samplesat a 1:1 ratio for 6 days in the presence of IL-7 and IL-15 (10 ng/mL).Cells were then labeled with antibodies against CD3 and the anti-CD19CAR and analyzed by flow cytometry (FIG. 34B). Cells that were mockelectroporated and mock transduced showed low levels of backgroundstaining in the CD3⁻/CAR⁺ quadrant (0.2%). The CD3⁻/CAR⁺ population wasthe same in samples that were mock electroporated then transduced withAAV (0.2%) and slightly higher in cells that were electroporated withTRC 1-2x.87EE and mock transduced (1.24%). The increase in CD3⁻/CAR⁺cells the TRC 1-2x.87EE alone control is considered background since noCAR nucleic acid was ever introduced into the system. Cells that wereelectroporated with TRC 1-2x.87EE mRNA and mock transduced had aCD3⁻/CAR⁻ population of 42.5%, which is significantly lower than theywere prior to expansion (56.7%, FIG. 33) suggesting that CD+ cells mayhave a growth advantage in this system. However, cells that wereelectroporated with TRC 1-2x.87EE and transduced with AAV showed 49.9%CD3⁻/CAR⁺ cells, a dramatic increase compared to the original labelingof these cells shown in FIG. 33 (13.4%), demonstrating that incubationof this sample with IM-9 cells in the presence of IL-7 and IL-15 isquite effective in enriching and expanding the CD3⁻/CAR⁺ population. TheCD3+/CAR⁺ population was also expanded under these conditions, with themock electroporated/AAV transduced sample and the TRC 1-2x.87EEelectroporated/AV transduced sample showing 2.53% and 15.3% CD3⁺/CAR⁺,respectively.

In the cells that were electroporated with TRC 1-2x.87EE then transducedwith AAV421, 24.2% of the CD3⁻ population was CAR⁺ prior to expansion(FIG. 33B). After incubation in medium supplemented with IL-7 and IL-15,that 25.3% of the CD3⁻ cells were CAR⁺ (FIG. 34A) indicating that theratio of gene knock-in to gene-knockout was unchanged. However, afterincubation with IM-9 cells in addition to IL-7 and IL-15, over 80%(80.35%, FIG. 34B) of the CD3⁻ cells were CAR⁺, demonstrating thatincubation with IM-9 cells resulted in antigen-specific enrichment.

Since mitocmyin C inactives cells very potently and IM-9 cells were notpersisting long in the mixed culture, we reasoned that a second infusionof IM-9 cells might further increase enrichment of CD3⁻/CAR⁺ cells. Someof the cells described above and shown in FIG. 34B would mixed withfresh IM-9 cells (pre-treated with mitocmycin C) in medium containingIL-7 and IL-15 and were incubated another 6 days. Cells were thenstained for CD3 and anti-CD19 CAR and analyzed by flow cytometry (FIG.34C). The percentage of CD3⁻/CAR⁺ cells in any of the control sampleswere essentially unchanged compared to the first round of enrichment onIM-9 cells.

However, the cells that were electroporated with TRC 1-2x.87EE andtransduced with AAV421 showed a significant enrichment of the CD3⁻/CAR⁺population, increasing from 49.9% (after the first round if incubationwith IM-9 cells, FIG. 34B) to 65.7% (FIG. 34C). Importantly, 93.75% ofthe CD3⁻ population was CAR⁺, indicating further antigen-specificexpansion.

3. Expression of Anti-CD19 Chimeric Antigen Receptor on T Cells UsingAAV422

We also examined expression of the anti-CD19 CAR from cells in whichAAV422 was used to provide the HDR template (described above, PCRresults shown in FIG. 32B). Approximately 4 days post-transduction,cells were labeled with antibodies that recognize the anti-CD19 CAR orCD3 and analyzed by flow cytometry. FIG. 35A shows flow cytometry plotsfor cells that were mock electroporated and transduced with increasingamounts of AAV422, along with control cells that were mockelectroporated and mock transduced. Cells that were mock electroporatedand mock transduced (MOI-0) were overwhelmingly CD3⁺/CAR⁻ (the lowerright quadrant, 98.8%). Cells that were mock electroporated thentransduced with increasing amounts of AAV422 looked essentiallyidentical to the control cells, with the CD3⁺/CAR⁻ populations at 98.6%,98.6%, 98.9% and 98.4%. Thus, the AAV422 vector alone is not drivingdetectable levels of CAR expression, nor is it capable of disruptingexpression of the T cell receptor.

FIG. 35B shows flow cytometry plots for samples that were eitherelectroporated with TRC 1-2x.87EE mRNA then mock transduced or cellsthat were electroporated with TRC 1-2x.87EE then transduced withincreasing amounts of AAV422. Cells that were electroporated then mocktransduced show 59.3% CD3⁻ cells, indicating efficient knockout of the Tcell receptor complex. Background labeling with the anti-CD19 CAR wasvery low, with 1.47% in the CD3⁻ population and 0.52% in the CD3⁺population. Samples that were electroporated with mRNA encoding TRC1-2x.87EE then transduced with increasing amounts of AAV422 showedsignificant amounts of CAR labeling in the CD3⁻ population, ranging from14.7% to 20.3%. There was also a slight increase in CAR labeling in theCD3⁺ population, ranging from 2.3% to 2.7%.

Surprisingly, we observed a noticeable increase in T cell receptorknockout efficiency in the presence of AAV422. Overall CD3 knockoutefficiency with increasing AAV422 was 71.6%, 74.9%, 77.8% and 74.4%compared to 59.3% in the TRC 1-2x.87EE electroporation alone. Incontrast, overall CD3 knockout efficiency with increasing AAV421 was56.99%, 56.62%, 57.4% and 55.4% compared to 57.18% in the TRC 1-2x.87EEelectroporation alone (FIG. 33B). Thus, it appears that electroporationwith TRC 1-2x.87EE in the presence of single-stranded AAV genomes, butnot self-complimentary AAV genomes, results in an increase in theoverall knockout efficiency of the TRC 1-2x.87EE nuclease. Because ofthis increase, the percent of CD3⁻ cells that are CAR′ is notsignificantly different between cells transduced with AAV421 and AAV422despite the higher numbers of CD3⁻/CAR⁺ cells. The highest percent ofCD3⁻ cells that were CAR′ using AAV421 was 24.18% (MOI=3.13e⁴ viralgenomes/cell) compared to 26.48% with AAV422 (MOI=1e⁵ viralgenomes/cell). This observation is particularly interesting consideringthe large difference in MOI between AAV421 and AAV422.

The concept of utilizing IM-9 cells to specifically enrich for CD3⁻/CAR⁺cells was tested using cells from this experiment. Again, rather thantesting the entire panel, we only attempted enrichment of either cellsmock electroporated then transduced with AAV422 or electroporated withTRC 1-2x.87EE then transduced with AAV422 (2.5e⁴ viral genomes/cell) ina new experiment. FIG. 36A shows flow cytometry plots at approximatelyday 4 post-transduction. Mock electroporated/transduced cells showedbackground staining of CD3⁻/CAR⁺ cells at 0.13%. In comparison, cellselectroporated with TRC 1-2x.87EE the transduced with AAV422 showed4.44% CD3⁻/CAR⁺ cells. Cells were incubated with IM-9 cells (pre-treatedwith mitomycin) in the presence of IL-7 and IL-15 for 6 days asdescribed above, then analyzed by flow cytometry. FIG. 36B shows thatincubation with IM-9 cells dramatically increased the CD3⁻/CAR⁺population in AAV422 transduced cells to 35.8%. The CAR⁺ cells make up45.2% of the total CD3⁻ population, compared to 6.69% prior toenrichment (FIG. 36A). As above, we also further enriched by a secondaddition of IM-9 cells (FIG. 36C). Two rounds of incubation with IM-9cells resulted in 65.1% CD3⁻/CAR⁺ cells. The CAR⁺ cells make up 78.25%of the total CD3⁻ population, indicating significant, antigen-dependentenrichment of CD3⁻/CAR⁺ cells.

These data, in conjunction with the data presented above, clearlydemonstrate that cells that have had an anti-CD19 CAR gene inserted intothe TRC 1-2 recognition sequence can be successfully enriched byincubation with IM-9 cells in the presence of IL-7 and IL-15, and canresult in a CD3⁻ population that is over 90% CARP in as little as 12days of culture.

4. Increased Knockout Efficiency Observed when Using Single-Strand AAVVectors

In the present study, we followed up on the observation thatsingle-stranded AAV vectors increased knockout efficiency of the TRC1-2x.87EE nuclease. In a first experiment, cells were electroporatedwith TRC 1-2x.87EE (2 μg) and either mock transduced or transduced withincreasing amounts of AAV412 (6.25e⁴, 1.25e⁴, 2.5e⁴ or 5e⁴ viralgenomes/cell). On day 4 post-transduction, cells were labeled with anantibody against CD3 and analyzed by flow cytometry (FIG. 37A). In themock transduced cells, 20.7% are CD3⁻ compared to 21.6%, 23.7%, 25.5%and 25% with increasing AAV412, indicating that TRC 1-2x.87EE knockoutefficiency is up to 23% higher in the presence of AAV412 (25.5% comparedto 20.7%).

To determine whether this increase in knockout efficiency was nucleasespecific, in an additional experiment, cells were electroporated withmRNA (2 μg) encoding a nuclease targeting the β2-microglobulin gene andeither mock transduced or transduced with increasing amounts of AAV412.Cells were stained for β2-microglobulin on day 4 post-transduction andanalyzed by flow cytometry (FIG. 37B). In the mock transduced cells,β2-microglobulin knockout efficiency was 64.5% and increased in thetransduced cells to 68.6%, 70.7%, 77.2% and 82.5% with increasingamounts of AAV412, demonstrating an increase in knockout efficiency ofup to 27.9% (82.5% compared to 64.5%).

In a parallel experiment, cells were electroporated with TRC 1-2x.87EEmRNA and either mock transduced or transduced with AAV422 (using thesame MOIs as AAV412). Cells were labeled with an antibody against CD3and cells were analyzed by flow cytometry (FIG. 37C). The mocktransduced cells showed 62.2% T cell receptor knockout, and withincreasing amounts of AAV, the T cell receptor knockout frequencyincreased to 72.6%, 75.5%, 78.3% and 75.1%. Here, the presence of AAV422increases the knockout efficiency of TRC 1-2x.87EE by up to 25.8% (78.3%compared to 62.2%). It is striking that the increase in percent knockoutefficiency is almost identical between these three experiments, usingtwo different nucleases and two different AAV vectors. Taken together,these data strongly indicate that transduction of cells with singlestrand AAV vectors increase the knockout efficiency of our nucleases,irrespective of nuclease or AAV cargo.

5. Activity of T Cells Expressing Anti-CD19 Chimeric Antigen Receptor

The above experiments clearly demonstrate the generation of CAR T cellsby electroporating cells with TRC 1-2x.87EE mRNA, then immediatelytransducing cells with AAV421, and that these cells can be enriched fora CD3⁻/CAR⁺ population by co-culture with CD19 expressing IM-9 cells. Wenext examined the activity of these CAR T cells against target cells. Inthe first experiment, the cells described above and shown in FIG. 34Cwere used in an IFN-gamma ELISPOT assay, in which either CD19⁺ Rajicells or CD19⁻ U937 cells were the target population. As shown in FIG.38A, when anti-CD19 CAR T cells were incubated with U937 cells, they didnot secrete IFN-gamma regardless of the target:effector ratio.Incubating CAR T cells with Raji cells, however, resulted in high levelsof IFN-gamma secretion, in a dose-dependent manner, indicating thatsecretion of IFN-gamma is antigen-specific.

These CAR T cells were also used in a cell killing assay in whichluciferase-labeled Raji cells were the target. Briefly, CAR T cells wereincubated with luciferase-labeled Raji cells at a ratio of 10:1. Atseveral time points, cells were washed and lysed to measure luciferaseactivity as a measure of how many cells remained. Control cells showedluciferase activity greater than 5500 arbitrary units (FIG. 38B).Co-incubation for 2, 3, 4 and 5 hours resulted in a decrease inluciferase activity to 4598, 3292, 2750 and 1932 arbitrary units,respectively. Thus, within 5 hours of co-incubation, luciferase activitywas reduced approximately 65%, indicating strong cytolytic activity ofthe CAR T cells.

Taken together, these data demonstrate that anti-CD19 CAR T cellsgenerated according to the methods described herein are effective atkilling CD19+ cells.

Example 7 Linearized Plasmid DNA

1. Expression of Chimeric Antigen Receptor from Linearized Plasmid DNA

Since HDR templates produced by AAV are linear DNA molecules, wehypothesized that linear DNA from any source may be a suitable HDRtemplate for inserting a CAR gene into the TRC 1-2 recognition sequence.To test this, we generated several plasmids that contain an anti-CD19CAR gene flanked by homology arms that are homologous to the TRC 1-2recognition sequence locus. Different promoters were used in someplasmids, and homology arms were either “short” (200 bp on the 5′homology arm and 180 bp on the 3′ homology arm) to mimic theself-complimentary AAV vectors, or “long” (985 bp on the 5′ homology armand 763 bp on the 3′ homology arm) to mimic the single strand AAVvectors. Plasmids with short homology arms are labeled “pDS” and thosewith long homology arms are labeled “pDI.” Additionally, some plasmidcontained an intron upstream of the CAR gene.

The CAR donor plasmids were linearized at a restriction site in thevector backbone and gel purified. Human CD3⁺ T cells were eitherelectroporated with the linearized CAR donor plasmid alone (varyingamounts between 500 ng and 1000 ng, depending on the concentration ofthe purified linearized plasmid), or co-electroporated with TRC 1-2.87EEmRNA (2 μg). As controls, cells were either mock electroporated orelectroporated with TRC 1-2x.87EE alone. The graphs in FIG. 39 arelabelled with descriptions for all electroporations. Approximately 4days post-electroporation, cells were labelled with antibodies againstCD3 and the anti-CD19 CAR and analyzed by flow cytometry (FIG. 39). FIG.39A shows background CD3⁻/CAR⁺ staining of 0.15%. It should be notedthat the background CD3⁺/CAR⁺ staining was unusually high at 4.31%. FIG.39B shows cells that were electroporated with TRC 1-2x.87EE mRNA alone,demonstrating 60.8% CD3 knockout. FIGS. 39C and 39D represent samplesthat were co-electroporated with TRC 1-2x.87EE mRNA and either the longhomology arm vector with an EF1α core promoter with an HTLV enhancer orthe short homology arm vector with EF1α core promoter (with noenhancer). Interestingly, the linearized CAR donor with the EF1α corepromoter alone generated a CD3⁻/CAR⁺ population of 2.38%, while thevector harboring the EF1α core promoter with the HTLV enhancer did notgenerate a significant percentage of CD3⁻/CAR⁺ cells. Cells that wereelectroporated with these two vectors in the absence of TRC 1-2x.87EEmRNA showed no significant increase in the CD3⁻/CAR⁺ population (FIGS.39E and 39F). The increase in the CD3⁻/CAR⁺ population with the EF1αcore promoter vector in the presence of TRC 1-2x.87EE suggested that alinearized plasmid could serve as an HDR template to repair doublestrand breaks at the TRC 1-2 recognition sequence.

FIGS. 39G and 39H show two long homology arm constructs that bothcontain an MND promoter driving expression of the CAR. One of theseconstructs, shown in FIG. 39G, also contains an intron in the 5′ end ofthe CAR gene. Surprisingly, the long homology arm plasmid with an MNDpromoter and intron showed significant CAR expression (FIG. 39G, 4.14%CD3⁻/CAR⁺) while the intron-less construct (FIG. 39H) did not showdetectable CAR expression when co-electroporated with TRC 1-2x.87EEmRNA. A short homology arm plasmid with the MND promoter, but with nointron, was also tested with TRC 1-2x.87EE mRNA and did not demonstrateany CAR expression (FIG. 39I). None of the MND promoter-containingconstructs generated any CARP cells in the absence of TRC 1-2x.87EE mRNA(FIGS. 39J, 39K, and 39L).

Lastly in this experiment, we tested a short homology arm construct thatcontained a JeT promoter driving expression of the CAR and a “long”homology arm construct with a CMV promoter driving expression of theCAR. Alone, neither of these linearized plasmids resulted in significantCARP cells (FIGS. 390 and 39P). When cells were co-electroporated withTRC 1-2x.87EE mRNA, the JeT containing construct showed 2.69% CD3⁻CAR⁺cells and the CMV containing construct yielded 2.7% CD3⁻/CAR⁺ cells.

The flow plots shown in FIG. 39 clearly demonstrate that linearizedplasmid DNA that encodes the CAR, flanked by homology arms, can serve asHDR templates to repair DNA breaks caused by TRC 1-2x.87EE, resulting ininsertion of the CAR nucleic acid. It is clear that promoter strengthplays a significant role in expression of the CAR, and some promotersdrive more efficient expression when there is an intron in the gene.

To confirm that insertion of the CAR using linearized DNA constructs wasspecific to the TRC 1-2 recognition sequence locus, we analyzed cells asdescribed above using primers that sat within the CAR and outside of thehomology arms (FIG. 40, Table 11). Samples 1 and 2 are PCR products fromcells that were either mock electroporated or electroporated with onlymRNA encoding TRC 1-2x.87EE. Consistent with results shown above, no PCRbands are present indicating the lack of CAR gene in the TRC 1-2recognition site. Samples 3, 4 and 5 are from cells that wereco-electroporated with TRC 1-2x.87EE and a linearized CAR homologyplasmid (samples names in FIG. 40). Each sample shows two PCR bands ofthe predicted size indicating insertion of the CAR gene expressioncassette into the TRC 1-2 recognition site. Samples 6, 7, and 8 are fromcells that were electroporated with the same linearized CAR homologyplasmids as samples 3, 4, and 5 but without TRC 1-2x.87EE mRNA. Asexpected, no PCR bands are present. Samples 9 and 10 are PCR productsfrom cells that were either mock electroporated or electroporated withonly mRNA encoding TRC 1-2x.87EE and show no PCR bands. Samples 11, 12,13 and 14 are from cells that were co-electroporated with TRC 1-2x.87EEand a linearized CAR homology plasmid (samples names in FIG. 40). Eachsample shows two PCR bands of the predicted size indicating insertion ofthe CAR gene into the TRC 1-2 recognition site. Samples 15, 16, 17, and18 are from cells that were electroporated with the same linearized CARhomology plasmids as samples 11, 12, 13, and 14 but without TRC1-2x.87EE mRNA. As expected, no PCR bands are present.

FIGS. 39 and 40 clearly demonstrate that co-electroporating human CD3⁺ Tcells with mRNA encoding TRC 1-2x.87EE and a linearized CAR homologyplasmid is an effective method to insert the CAR gene into the TRC 1-2recognition sequence.

TABLE 11 Linearized Sample Nucleofection plasmid  1 Mock (Water) —  2TRC1-2x87EE —  3 TRC1-2x87EE pDS EF1-α Core  4 TRC1-2x87EE pDS 200 MNDNC  5 TRC1-2x87EE pDS 200 JET NC  6 Mock (Water) pDS EF1-α Core  7 Mock(Water) pDS 200 MND NC  8 Mock (Water) pDS 200 JET NC  9 Mock (Water) —10 TRC1-2x87EE — 11 TRC1-2x87EE pDI EF1-α NC 12 TRC1-2x87EE pDI MNDintron NC 13 TRC1-2x87EE pDI MND NC 14 TRC1-2x87EE pDI CMV 985 NC 763 15Mock (Water) pDI EF1-α NC 16 Mock (Water) pDI MND intron NC 17 Mock(Water) pDI MND NC 18 Mock (Water) pDI CMV 985 NC 763 19 Mock (Water) —20 TRC1-2x87EE — 21 TRC1-2x87EE pDS MCS 22 Mock (Water) PDS MCS

Example 8 Characterization of Additional AAV Vectors

1. Use of AAV with JeT Promoter and Long Homology Arms

Collectively, the data shown above indicate that vectors utilizing theJeT promoter drive high, consistent expression of the CAR and thatlonger homology arms may increase gene insertion efficiency. We designedand generated the vector shown in FIG. 41 (SEQ ID NO:125), which wasused to make single-strand AAV with long homology arms, and a JeTpromoter driving expression of the anti-CD19 CAR (referred to herein asAAV423). Human CD3⁺ T cells were electroporated with mRNA encoding TRC1-2x.87EE and transduced with increasing amounts of AAV423. Since datashown above suggested that higher MOIs may result in increased insertionefficiency, we used titers ranging from 1.875e⁴ to 1.5e⁵. As controls,cells were either electroporated with mRNA encoding TRC 1-2x.87EE thenmock transduced or mock electroporated then transduced with increasingamounts of AAV423. On day 6 post-transduction, cells were labeled withantibodies recognizing CD3 or the anti-CD19 CAR and analyzed by flowcytometry. As shown in FIG. 42, cells that were mock electroporated thentransduced with increasing amounts of AAV423 are overwhelminglyCD3⁺/CAR⁻ (ranging from 96.6% to 98.5%). Cells that were electroporatedwith mRNA encoding TRC 1-2x.87EE and mock transduced were 39% CD3⁻indicating efficient knockout of the T cell receptor. In these cells,background CAR staining was very low (around 2%). Cells that wereelectroporated with mRNA encoding TRC 1-2x.87EE then transduced withincreasing amounts of AAV423 showed dramatic CAR staining in conjunctionwith CD3 knockout. CD3⁻/CAR⁺ populations ranged from 21.6% to 22.7%,while CD3⁺/CAR⁺ populations were around 2%. As described above, thepresence of single-strand AAV increased the overall gene modificationefficiency at the TRC 1-2 recognition site, with total CD3⁻ populationsincreasing from 41.44% in the control cells to 57.6%, 59.2%, 58.7%, and56.1% in cells that were electroporated then transduced with increasingamounts of AV423. The percent of CD3⁻ cells that were CARP ranged from37.5% to 39.9% indicating a dramatic increase in insertion efficiencycompared to data described above.

To confirm that insertion of the CAR using AAV423 was specific to theTRC 1-2 recognition sequence locus, we analyzed cells as described aboveusing primers that sat within the CAR and outside of the homology arms(FIG. 43, Table 12).

TABLE 12 Sample Nucleofection AAV (μl) MOI  1 Mock (Water) — —  2 Mock(Water) — —  3 Mock (Water) pDI JET Prep A (3.125) 18750  4 Mock (Water)pDI JET Prep A (6.25) 37500  5 Mock (Water) pDI JET Prep A (12.5) 7500 6 Mock (Water) pDI JET Prep A (25) 150000  7 TRC1-2x87EE — —  8TRC1-2x87EE pDI JET Prep A (3.125) 18750  9 TRC1-2x87EE pDI JET Prep A(6.25) 37500 10 TRC1-2x87EE pDI JET Prep A (12.5) 7500 11 TRC1-2x87EEpDI JET Prep A (25) 150000Samples 1 and 2 are PCR products from cells that were mockelectroporated. Consistent with results shown above, no PCR bands arepresent indicating the lack of CAR gene in the TRC 1-2 recognition site.Samples 3-6 are from cells that were mock electroporated then transducedwith increasing amounts of AAV423. Consistent with results above, thereare no PCR bands present. Sample 7 is from cells electroporated withmRNA encoding TRC 1-2x.87EE then mock transduced, and shows no PCRbands. Samples 8-11 are from cells electroporated with mRNA encoding TRC1-2x.87EE then transduced with increasing amounts of AAV423, and showthe PCR bands expected if the CAR is inserted into the TRC 1-2recognition sequence.

Given the ability of AAV423 to insert the CAR sequence into the TRC 1-2recognition site following cleavage, it is further envisioned that theAAV423 plasmid (FIG. 41) could be linearized by digestion with a anddelivered to the cell by digestion with one or more restriction enzymes,such that the T cells could be transfected with a linearized DNAtemplate which could integrate into the TRC 1-2 recognition site andencode an anti-CD19 CAR.

Example 9 In Vivo Efficacy of Anti-CD19 TCR-Negative CAR T Cells 1.Murine Model of Disseminated B Cell Lymphoma

The efficacy of the gene-edited anti-CD19 CAR T cells was evaluated in amurine model of disseminated B cell lymphoma. Activated T cells wereelectroporated with TRC 1-2x.87 EE mRNA as described above, thentransduced with an AAV6 vector comprising an anti-CD19 CAR expressioncassette driven by a JeT promoter and flanked by homology arms.Following 5 days of culture with IL-2 (10 ng/mL), cells were analyzedfor cell-surface CD3 and anti-CD19 CAR expression by flow cytometry aspreviously described (FIG. 44A). CD3⁻ cells were enriched by depletingCD3⁺ cells using anti-CD3 magnetic beads. Depleted cells were thencultured for 3 days in IL-15 (10 ng/mL) and IL-21 (10 ng/mL) andre-analyzed for cell-surface expression of CD3 and anti-CD19 CAR (FIG.44B). Isolation of the CD3⁻ population was quite efficient, yielding99.9% purity as measured by flow cytometry following depletion of CD3⁺cells (FIG. 44B). The purified CD3⁻ population comprised 56% CD4+ and44% CD8⁺ cells (FIG. 44C), and had primarily central memory/transitionalmemory phenotypes, determined by staining for CD62L and CD45RO (FIG.44D).

Studies utilizing the Raji disseminated lymphoma model were conducted byCharles River Laboratories International Inc. (Morrisville, N.C., USA).CD19⁺ Raji cells stably expressing firefly luciferase (ffLuc)⁴⁴ wereinjected i.v. into 5-6 week old female NSG mice on Day 1, at a dose of2.0×10⁵ cells per mouse. On Day 4 mice were injected i.v. with PBS orPBS containing gene edited control TCR KO T cells prepared from the samehealthy donor PBMC or PBS containing the indicated doses of CAR T cellsprepared from the same donor. On the indicated days, live mice wereinjected i.p. with Luciferin substrate (150 mg/kg in saline),anesthetized, and Luciferase activity measured after 7 minutes usingIVIS SpectrumCT (Perkin Elmer, Waltham, Mass.). Data was analyzed andexported using Living Image software 4.5.1 (Perkin Elmer, Waltham,Mass.). Luminescence signal intensity is represented by radiance inp/sec/cm²/sr.

2. Results

As shown in FIG. 45, growth of CD19⁺ Raji cells was evident in all miceat low levels by day 8, and increased significantly in untreated andTCR⁻ control groups by day 11. In control groups, significant tumorgrowth was observed by day 15, and by day 18 or 19 all control groupswere euthanized. In contrast, all groups of mice treated with anti-CD19CAR T cells showed no evidence of tumor growth by day 11 and, with theexception of a single mouse in the low dose group, remained tumor-freethrough day 29 of the study. Tumor re-growth was observed in three micein the low dose cohort around day 36. One of the three died at day 42,though imaging revealed only low levels of tumor in this animal, so itis unlikely that death was tumor-related.

3. Conclusions

These results provide clear evidence for in vivo clearance of CD19⁺tumor cells by gene-edited CD3⁻ CAR T cells and support furtherpreclinical development of this platform for allogeneic CAR T celltherapy.

1-9. (canceled)
 10. A method for producing a genetically-modifiedeukaryotic cell by disrupting a target sequence in a chromosome of saideukaryotic cell, said method comprising: transfecting a eukaryotic cellwith a nucleic acid encoding an engineered meganuclease, wherein saidengineered meganuclease binds and cleaves at a recognition sequenceconsisting of SEQ ID NO: 3, wherein said engineered meganucleasecomprises a first subunit and a second subunit, wherein said firstsubunit binds to a first recognition half-site of said recognitionsequence, and wherein said second subunit binds to a second recognitionhalf-site of said recognition sequence, and wherein said engineeredmeganuclease comprises an amino acid sequence having at least 96%identity to SEQ ID NO: 8; wherein said engineered meganuclease producesa cleavage site in said chromosome at a recognition sequence consistingof SEQ ID NO: 3, and wherein said target sequence is disrupted bynon-homologous end-joining at said cleavage site.
 11. The method ofclaim 10, wherein said eukaryotic cell is a human T cell or a cellderived therefrom.
 12. The method of claim 10 further comprisingtransfecting said eukaryotic cell with a second nucleic acid includingan exogenous sequence of interest.
 13. The method of claim 12, whereinsaid second nucleic acid further comprises sequences homologous tosequences flanking said cleavage site, and wherein said exogenoussequence of interest is inserted at said cleavage site by homologousrecombination.
 14. The method of claim 12, wherein said sequence ofinterest encodes a chimeric antigen receptor.
 15. The method of claim12, wherein at least said second nucleic acid is introduced into saideukaryotic cell by a recombinant AAV vector.
 16. A method for producinga genetically-modified eukaryotic cell by disrupting a target sequencein a chromosome of said eukaryotic cell, comprising: introducing anengineered meganuclease into a eukaryotic cell, wherein said engineeredmeganuclease binds and cleaves at a recognition sequence consisting ofSEQ ID NO: 3, wherein said engineered meganuclease comprises a firstsubunit and a second subunit, wherein said first subunit binds to afirst recognition half-site of said recognition sequence, wherein saidsecond subunit binds to a second recognition half-site of saidrecognition sequence, and wherein said engineered meganuclease comprisesan amino acid sequence having at least 96% identity to SEQ ID NO: 8;wherein said meganuclease produces a cleavage site in said chromosome ata recognition sequence consisting of SEQ ID NO: 3, and wherein saidtarget sequence is disrupted by non-homologous end-joining at saidcleavage site.
 17. The method of claim 16, wherein said eukaryotic cellis a human T cell or a cell derived therefrom.
 18. The method of claim16 further comprising transfecting said eukaryotic cell with a nucleicacid including an exogenous sequence of interest.
 19. The method ofclaim 18, wherein said nucleic acid further comprises sequenceshomologous to sequences flanking said cleavage site, and wherein saidexogenous sequence of interest is inserted at said cleavage site byhomologous recombination.
 20. The method of claim 18, wherein saidsequence of interest encodes a chimeric antigen receptor.
 21. The methodof claim 18, wherein said nucleic acid is introduced into saideukaryotic cell by a recombinant AAV vector.
 22. A method ofimmunotherapy for treating cancer in a subject in need thereof, saidmethod comprising administering to said subject a pharmaceuticalcomposition comprising said genetically-modified cell produced by themethod of claim 1 and a pharmaceutically acceptable carrier.
 23. Themethod of claim 22, wherein said cancer is selected from the groupconsisting of carcinoma, lymphoma, sarcoma, blastomas, and leukemia. 24.The method of claim 22, wherein the cancer is selected from the groupconsisting of a cancer of B-cell origin, breast cancer, gastric cancer,neuroblastoma, osteosarcoma, lung cancer, melanoma, prostate cancer,colon cancer, renal cell carcinoma, ovarian cancer, rhabdomyosarcoma,leukemia, and Hodgkin's lymphoma.
 25. The method of claim 24, whereinthe cancer of B-cell origin is selected from the group consisting ofB-lineage acute lymphoblastic leukemia, B-cell chronic lymphocyticleukemia, and B-cell non-Hodgkin's lymphoma.
 26. A use of an engineeredmeganuclease having at least 96% identity to SEQ ID NO: 8 to treat asubject in need of treatment thereof.
 27. The use of claim 26, whereinsaid subject has a cancer that is selected from the group consisting ofcarcinoma, lymphoma, sarcoma, blastomas, and leukemia.
 28. The use ofclaim 26, wherein the cancer is selected from the group consisting of acancer of B-cell origin, breast cancer, gastric cancer, neuroblastoma,osteosarcoma, lung cancer, melanoma, prostate cancer, colon cancer,renal cell carcinoma, ovarian cancer, rhabdomyosarcoma, leukemia, andHodgkin's lymphoma.
 29. The use of claim 28, wherein the cancer ofB-cell origin is selected from the group consisting of B-lineage acutelymphoblastic leukemia, B-cell chronic lymphocytic leukemia, and B-cellnon-Hodgkin's lymphoma.